Method for detecting replication or colonization of a biological therapeutic

ABSTRACT

Methods for detecting replication in or colonization of a host by a biological therapeutic, such as an oncolytic virus, cells administered for cell therapy and gene therapy vectors, are provided. In the methods, a product produced by the biological therapeutic is detected in a sample of tissue or body fluid distinct from the administered therapy or locus thereof, thereby permitting assessment of the therapy and/or monitoring its progress.

RELATED APPLICATIONS

This application is a continuation of now allowed co-pending U.S. patentapplication Ser. No. 13/573,845, filed Oct. 5, 2012, entitled “Methodfor Detecting Replication or Colonization of a Biological Therapeutic,”to Aladar A. Szalay, Jochen Stritzker, and Michael Hess, which claimsbenefit of priority to U.S. provisional Application No. 61/627,255,filed Oct. 5, 2011, entitled “Method for Detecting Replication orColonization of a Biological Therapeutic.”

This application is related to corresponding International ApplicationNo. PCT/US2012/059126, filed Oct. 5, 2012, entitled “Method forDetecting Replication or Colonization of a Biological Therapeutic,”which claims priority to U.S. provisional Application No. 61/627,255,filed Oct. 5, 2011.

The subject matter of each of the above-noted applications isincorporated by reference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED ELECTRONICALLY

An electronic version of the Sequence Listing is filed herewith, thecontents of which are incorporated by reference in their entirety. Theelectronic file was created on Sep. 26, 2014, is 3,181 kilobytes insize, and titled 4838BSEQ001.txt.

APPLICATIONS INCORPORATED BY REFERENCE

The following applications and patents, which describe inter alia,viruses and bacteria and methods of preparing and using such viruses andbacteria, are incorporated by reference: U.S. Pat. Nos. 7,588,767,7,588,771, 7,662,398, 7,754,221, 7,763,420, 7,820,184, 8,021,662,8,052,968, 8,066,984, 8,221,769 and U.S. Patent Publication Nos.2011/0300176, 2007/0202572, 2007/0212727, 2010/0062016, 2009/0098529,2009/0053244, 2009/0155287, 2009/0117034, 2010/0233078, 2009/0162288,2010/0196325, 2009/0136917, 2011/0064650, 2003/0059400, 2004/0234455,2005/0069491, 2009/0117049, 2009/0117048, 2009/0117047, 2009/0123382,2003/0228261, 2004/0213741 and 2005/0249670 and U.S. patent applicationSer. No. 13/506,369.

FIELD OF THE INVENTION

Methods for detecting replication or colonization of a biologicaltherapeutic are provided.

BACKGROUND

Biological therapies, such as gene therapies, cell therapies andoncolytic viral therapies, are viable treatment modalities. Monitoringtheir administration and effectiveness, however, is difficult. Hence,there exists a need for a facile, generally applicable method fordetecting or assessing, gene expression, cell colonization or infectiondetermining tumor colonization and/or gene expression in subjectstreated with biological therapies. These and other needs are addressedherein.

SUMMARY

Provided are methods for detecting replication in a subject and/orcolonization of a target locus in the subject by a biologicaltherapeutic, such as a therapeutic virus, or detecting replication ofthe biological therapeutic in a cell the subject by detecting thepresence of a protein that is encoded by the biological therapeuticseparate from the biological therapeutic or the cell in which it isexpressed. When a biological therapeutic, such as an oncolytic virus, isadministered, its ability to effect treatment depends upon its abilityto accumulate in target cells, such as tumor cells. For oncolyticviruses this requires that the virus colonize target cells and replicatetherein to lyse the target cells. Upon replication, gene expressionoccurs and virally encoded proteins are expressed. When the cells lyseor otherwise release proteins, the virally encoded proteins or productsthereof appear in body fluids, such as blood, plasma and urine. Thevirally encoded proteins and products are not necessarily secretedproducts, but they appear in body fluids because cells have lysed. Themethods herein detect such proteins and products.

The methods herein are practiced by obtaining a sample from a subject towhom the therapeutic has been administered; and then testing the sampleto detect the protein encoded by the biological therapeutic or a productthereof. Detection of the protein or product indicates that thebiological therapeutic is replicating in or colonizing a target locus.Generally the protein, when expressed, is not operatively linked to asignal sequence so that it is not secreted. The protein or product isreferred to herein as a reporter protein, and the nucleic acid encodingit is a reporter gene. The protein can be detected directly orindirectly. Presence of the protein in such a sample indicates that thebiological therapeutic is replicating and/or its genes are beingexpressed in the host to which it is administered. The sample generallyis obtained from a locus in the subject other than a target of thebiological therapeutic, unless, the target is tumor of such locus, suchas leukemia. Generally, the sample comprises a body fluid, or is asample that does not contain the biological therapeutic. When the samplecontains the biological therapeutic, testing is effected underconditions or is corrected or normalized, such that expression from thebiological therapeutic in the sample is not considered. The reporterprotein encoded by the biological therapeutic can be an endogenousprotein or a heterologous protein, and typically is a detectable proteinor is a protein, such as an enzyme, that can produce a detectablesignal. Reporter proteins include, but are not limited to, enzymes,antigens and enzyme substrates. Detection can include, for example,contacting the sample with a substrate if the reporter is an enzyme anddetecting a product catalyzed by the enzyme. Where the reporter proteinis an antigen, detection can be effected by capturing with an antibodyor other binding molecule, such as an antibody presented on ananoparticle.

Exemplary of the methods are methods of detecting tumors byadministering a pox virus, such as a vaccinia virus to a subject. Thevirus encodes an enzyme, such as a beta-glucuronidase, such as abacterial beta-glucuronidase; and the method then includes obtaining abody fluid sample, such as blood, urine, CSF or other body fluid; anddetecting beta-glucuronidase activity in the sample, where detection ofactivity indicates the presence of tumor cells in the subject. Themethod can detect the presence of tumors or tumor cells, such ascirculating tumor cells and metastasizing cells, in the subject and alsocan be employed to monitor tumor therapy, including therapy with theadministered vaccinia virus, which serves as a theranostic. The poxviruses, such as vaccinia viruses, include any known to those of skillin the art and any described herein.

Subjects include any subject, including but not limited to any animal.The animals include humans, veterinary animals, pets, farm animals,animal models. Included are human, cats, dogs, frogs, ferrets, gorillas,chimpanzees, gorillas, birds, lions, tigers, cows, rodents, goats, pigs,chickens, horses, zebras, dolphins and whales.

Biological therapeutics, include, but are not limited to, viruses,particularly oncolytic viruses, such as vaccinia viruses, gene therapyvectors, including viruses, cells for cell therapy, such as adoptiveimmunotherapy, transplanted cells, autologous cell therapy, stem celltherapy, bacteria, particularly non-pathogenic bacteria that accumulatein tumor cells and other immunoprivileged cells and tissues; nucleicacids in vectors or in delivery vehicles, such as targeted liposomes.Exemplary of viruses are oncolytic viruses, such as vaccinia viruses,particularly those that accumulate in immunoprivileged cells, such astumors. Such viruses include, vaccinia viruses derived from the listerstrain, such as LIVP viruses and derivatives thereof, including virusesmodified to include heterologous nucleic acid.

Oncolytic viruses include, but are not limited to, poxvirus, oncolyticadenovirus, reovirus, herpes virus, adeno-associated virus, lentivirus,retrovirus, rhabdovirus, papillomavirus, vesicular stomatitis virus,measles virus, Newcastle disease virus, picornavirus, sindbis virus,papillomavirus, parvovirus, coxsackievirus, influenza virus, mumpsvirus, poliovirus and semliki forest virus. In some instances, theviruses, such as adenoviruses, must be modified to be oncolytic (i.e.selectively replicate and ultimately lyse tumor cells). Poxvirusesinclude, but are not limited to viruses selected from amongorthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, molluscipoxvirus, yatapoxvirus, entomopoxvirus A,entomopoxvirus B and entomopoxvirus C.

Vaccinia viruses, such as Lister strain viruses, including LIVP viruses,including clonal variants and clonal isolates of such viruses arecontemplated for administration. Exemplary LIVP viruses and isolates andderivatives thereof include the virus designated GLV-1h68, describedherein and known to those of skill in the art, and derivatives (virusesthat are produced from GLV-1h68 or related viruses) and modified formthereof. Exemplary of such viruses are any whose genomes comprise asequence of nucleotides set forth in any of SEQ ID NOS:82-83 and 85-91,or a sequence of nucleotides that has at least 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more sequenceidentity to a sequence of nucleotides set forth in any of SEQ ID NOS:82-83 and 85-91. Exemplary of such viruses that are derivatives orrelated to GLV-1h68 as defined above, are those designated GLV-1h22,GLV-1i69, GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h80,GLV-1h81, GLV-1h82, GLV-1h83, GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87,GLV-1j88, GLV-1j89, GLV-1h90, GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97,GLV-1h98, GLV-1h99, GLV-1h100, GLV-1h101, GLV-1h104, GLV-1h105,GLV-1h106, GLV-1h107, GLV-1h108, GLV-1h109, GLV-1h139, GLV-1h143,GLV-1h146, GLV-1h150, GLV-1h151, GLV-1h152 and GLV-1h153. These virusescan be modified, if they do not express a reporter, to express a desiredreporter gene or protein, such as a glucuronidase. As shown herein,glucuronidase and products thereof result in a highly sensitive systemfor detecting as few as a single cell in a sample. Thus, such system canbe used for early detection of tumors, as well as for monitoringtherapy.

The oncolytic viruses accumulate in tumors, but as shown herein,non-tumor samples, such as urine, blood, serum and other body fluids canbe tested to detect the presence of viral encoded proteins. The presenceof the protein outside of the cells that contain the virus indicatesthat the virus has colonized the cells and is replicating. Encodedproteins are shed as the cells are lysed. Cells, such as tumors, wheninfected with the virus, become protein factors and can produce largeamounts of proteins. Hence, administration of a virus (or otherbiological therapeutic) can be assessed by detecting the presence ofencoded protein. As shown herein, the presence of tumors as small as 1mm³ or even smaller can be detected. Thus, the methods herein permitdiagnosis of tumors. The methods herein also permit detection ofcirculating tumor cells.

In addition, the methods can be used to assess the effectiveness oftherapy. Detection of a protein encoded by a biological therapeuticindicates that it is being expressed and/or replicating (depending uponthe therapeutic). Hence the methods can monitor treatment to discernwhether viral colonization of a target locus has been established.Therapy also can be monitored over time. The particular results todetect depend upon the type of therapy. For example, for tumor therapy,initially, there should be detectable protein expressed, but as tumorsare cleared and disappear the amount of protein will decrease. For genetherapy methods in which nucleic acids become part of the genome, longerperiods of expression may be expected, and instances permanent levels ofexpression may be expected. The methods herein permit monitoring of anysuch type of therapy. For example, subjects treated with such therapy,can be periodically monitored through blood or urine or other body fluidsamples.

The therapeutic virus can be administered topically, locally andsystemically, including enterally or parenterally. Such methods include,but are not limited to, administration that is effected orally,epicutaneously, intranasally, by gastric feeding tube, duodenal feedingtube, or gastrostomy, rectally, intravenously, intraarterially,intramuscularly, intracardiacly, subcutaneously, by intraosseousinfusion (into the bone marrow), intradermally, intraperitoneally,intrapleurally, transdermally, transmucosally, epidurally,intrathecally, intraventricularly or intratumorally.

The dosage administered and/or administration regimen depend upon thebiological therapeutic administered and its purpose. For the oncolyticvaccinia virus, including any described herein, dosages typicallyinclude at least or about 1×10⁵ or about 1×10⁵ plaque forming units(PFU), 5×10⁵ or about 5×10⁵ PFU, at least 1×10⁶ or about 1×10⁶ PFU,5×10⁶ or about 5×10⁶ PFU, 1×10⁷ or about 1×10⁷ PFU, 5×10⁷ or about 5×10⁷PFU, 1×10⁸ or about 1×10⁸ PFU, 5×10⁸ or about 5×10⁸ PFU, 1×10⁹ or about1×10⁹ PFU, 5×10⁹ or about 5×10⁹ PFU, 1×10¹⁰ or about 1×10¹⁰ PFU or5×10¹⁰ or about 5×10¹⁰ PFU as total single dosage for an average humanof 75 kg or adjusted for the weight and size and species of the subjectand the mode of administration. One of skill in the art can determinesuitable dosage. Suitable dosages for any biological therapeutic can bedetermined empirically.

Viruses and vectors administered include those selected from among aretrovirus, adenovirus, adeno-associated virus and herpes simplex virus.The vectors can be administered as naked nucleic acid or in a suitabledelivery vehicle, including, for example, a liposome, PEGylatedliposome, nanoparticle, lipid-based nanoparticle or lymphocyte.

The oncolytic viruses, such as vaccinia virus, and the bacteria, such asattenuated or non-pathogenic bacterium, accumulate in immunoprivilegedcells and tissues, which are cells and tissues, such as inflamed/woundedtissues, particularly inside a subject, and tumors, which tissues aresheltered or not exposed to the immune system, which clears the virusesand bacteria from other sites. Immunoprivileged cells and tissuesinclude sites of cellular proliferation, such as tumors, tumor tissues,metastases, areas of inflammation, wounds and infections. Bacteria,particularly attenuated or non-pathogenic bacteria, include a mutual,commensal or probiotic strain of bacteria or an attenuated pathogenicbacterium. Strains of bacteria include, but are not limited to bacteriaselected from among Escherichia coli, Bacteroides, Eubacterium,Streptococcus, Actinomyces, Veillonella, Nesseria, Prevotella,Campylobacter, Fusobacterium, Eikenella, Porphyromonas,Priopionibacteria, Clostridia, Salmonella, Shigella, Bifidobacteria andStaphylococcus species. Such bacteria include the probiotic E. colibacterium, Nissle, such as Nissle 1917.

Cell therapies include, but are not limited to, cell transplants, suchas, for example, cell transplants selected from among pancreatic islet,bone marrow, endothelial, epidermal, myoblast, neural and stem celltransplants. Biological therapies that include expression vectors andgene therapy vectors, include, but are not limited to, a viral vector,mammalian vector, bacterial vector, insect vector, plant vector orartificial chromosome encoding the reporter gene, such as retroviruses,satellite artificial chromosomes, baculoviruses, vaccinia viruses andothers well known to those of skill in the art. For cell therapies,detection can be effected by infecting or transfecting or transducingthe cell with nucleic acid encoding the reporter protein. The nucleicacid can be introduced on a vector, virus, bacterium or artificialchromosome including any listed elsewhere herein. Generally for celltherapy the nucleic acid is introduced prior to administration tosubject.

For practicing the methods, as noted, any protein encoded by thebiological therapeutic can be detected. These include enzyme orenzymatic reporter proteins (i.e., proteins that catalyze a reactionwhen contacted with a substrate). The nucleic acid encoding the enzyme,typically is not linked to nucleic acid encoding a secretory signal, butinstead occurs in tissues and body fluids of a subject upon shedding bya cell containing the biological therapeutic or the cell that is abiological therapeutic. For enzymes, typically, the sample or an aliquotof the sample or treated sample to adjust conditions or purify orpartially purify the reporter protein, is contacted with substrate forthe enzyme. A product that is produced by this reaction is detected.Exemplary of this are oncolytic viruses, such as vaccinia viruses, thatencode an enzyme, particularly a bacterial enzyme not normally expressedin the treated subject. In addition, the biological therapeutic can be acell or gene therapy vector or oncolytic virus; and detecting the shedreporter enzyme assesses the progress of cancer therapy, immunotherapy,adoptive immunotherapy or gene therapy administered in connection withcancer therapy, immunotherapy, adoptive immunotherapy or gene therapy.

For cancer therapy the tumors include solid tumors, blood and lymphaticcancers and metastases, including, but are not limited to tumorsselected from among a bladder tumor, breast tumor, prostate tumor,glioma tumor, adenocarcinoma; ovarian carcinoma, and pancreaticcarcinoma, liver tumor and skin tumor, pancreatic cancer, non-small celllung cancer, multiple myeloma, leukemia, lung and bronchus tumor, breasttumor, colon and rectum tumor, kidney tumor, stomach tumor, esophagustumor, liver and intrahepatic bile duct tumor, urinary bladder tumor,brain tumor and other nervous system tumor, head and neck tumor, oralcavity tumor and pharynx tumor, cervix tumor, uterine corpus tumor,thyroid tumor, ovary tumor, testes tumor, prostate tumor, malignantmelanoma, cholangiocarcinoma, thymoma, non-melanoma skin cancer;hematologic tumor, malignancy, childhood leukemia and lymphoma, multiplemyeloma, Hodgkin's disease, lymphomas of lymphocytic and cutaneousorigin, acute and chronic leukemia, acute lymphoblastic leukemia, acutemyelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasmor lymphoid neoplasm and cancers associated with AIDS. In addition,administered biological therapeutics include wounds and inflamed tissuesinside of a subject (not visible to the naked eye) that can be detectedor for which therapy is monitored.

As noted any protein encoded by the virus can be employed as a reporter.Typically such proteins include enzymes and antibodies and antigens andother readily detectable proteins or proteins that induce or produce adetectable signal. Detection, as noted, provides for detecting tumorsand also monitoring therapy with biological therapeutic. Such therapiesinclude oncolytic viral therapy, gene therapy, cell therapy,immunotherapy, adoptive immunotherapy or gene therapy. The protein to bedetected, the reporter protein, generally is operatively linked to apromoter. The promoter can be constitutive or inducible promoter orotherwise regulatable. The protein, such as those expressed by thevaccinia virus, can be linked to a secretory signal, but generally arenot, since the method detects shed proteins indicative, in the case ofoncolytic viruses, production of protein in tumor cells and shedding bysuch cells, typically by cell lysis or leakage.

As noted, the reporter gene can be an endogenous protein to the subjector to the biological therapeutic or can be heterologous and encoded bythe biological therapeutic. Included are eukaryotic and prokaryoticproteins, such as enzymes. Enzymes include, but are not limited to,enzymes selected from among lipases, phospholipases, sulfatases,ureases, peptidases, proteases, esterases, phosphatases, acidphosphatases, glycosidases, glucosidases, glucuronidases,galactosidases, carboxylesterases, luciferases, peroxidases, hydrolases,oxidoreductases, lyases, transferases, isomerases, ligases, synthases,protein kinases, esterases, isomerases, glycosylases, synthetases,dehydrogenases, oxidases, reductases, methylases, oxidases, P450enzymes, monoamine oxidases (MAOs), flavin monoamine oxidases (FMOs),transferases, glutathione S transferases (GSTs), alkaline phosphatases(AP), invertases, luciferases, acetyltransferases, beta-glucuronidases,exo-glucanases, glucoamylases, beta-glucosidases, horseradishperoxidases, alkaline phosphatases, beta-lactamases, alpha-amylases,alpha-glucosidases, catalases, beta-xylosidases, beta-galactosidases,chondroitinsulfatases, gelatinases, collagenases, caseinases,nitroreductases, azoreductases, demethylases, deacetylases,deformylases, phosphatases, kinases, peroxidases, sulfotases,acetylcholinesterases, dehydrogenases, dealkylases and oxygenases.Exemplary are β-glucuronidase, β-galactosidase, luciferase,chloramphenicol acetyltransferase (CAT) and alkaline phosphatase.

Enzymes are detected by contacting the sample with a suitable substratewhose product is detectable, such by a change in optical properties ofthe sample. Substrates can be fluorescent, luminescent,spectrophotometric, fluorogenic or chromogenic substrate or thesubstrate is a contrast agent or generates a contrast agent for PETimaging or generate products with such properties. Detection can beeffected by any detection method known to those of skill in the artsuitable for the particular product or change in property in a sample.Detection methods and properties, include, but are not limited to, PETimaging, MRI, mass spectrometry, and optical methods, such as methodsthat employ, for example, a spectrophotometer, fluorometer, luminometer,scintillation counter or a Raman spectrometer and other such devices.Products generated or substrates (whose conversion to product isdetectable) can be detected.

In the methods herein, the reporter protein, as noted, can be aβ-glucuronidase, including a mammalian, such as human, and bacterialβ-glucuronidase, such as one that includes a sequence of amino acids setforth in SEQ ID NO: 121 or SEQ ID NO:4, or an active portion thereof, oran enzyme that that exhibits at least 85% sequence identity to asequence of amino acids set forth in SEQ ID NO:121 or SEQ ID NO: 4 or anactive portion thereof. Other β-glucuronidase enzymes include any (oractive portions thereof) having at least 85% sequence identity theretoor to the active portion, include those whose sequences are set forth inany of SEQ ID NOS: 1-11, 114-121 and 127-146.

In the methods the enzymatic reporter protein can be a β-glucuronidase(as described herein), and the substrate is any suitable substrate,including but not limited to, any selected from among fluoresceindi-β-D-glucuronide (FDGlcU), methylumbelliferyl-β-D-glucuronide (4-MUG),carboxyumbelliferyl β-D-glucuronide (CUGlcU),5-(pentafluorobenzoylamino)fluorescein di-β-D-glucuronide (PFB-FDGlcU),C₁₂—Fluorescein β-D-Glucuronidase, 5-bromo-4-chloro-3-indolyl0-D-glucuronide (X-GlcU or BCIG), p-nitrophenyl-β-D-glucuronide,Red-β-D-GlcU,CHA (Magenta-b-D-GlcA;5-bromo-6-chloro-3-indolyl-b-D-glucuronide, cyclohexylammonium salt),Rose-β-D-GlcU,CHA (Salmon-β-D-GlcUA;5-bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt),phenyl-β-D-glucuronide, and suitable salts or other forms thereof. Forexample, the enzymatic reporter protein can β-glucuronidase and thesubstrate selected from among fluorescein di-β-D-glucuronide (FDGlcU)and 4-methylumbelliferyl-β-D-glucuronide (4-MUG). The β-glucuronidaseencoded by the virus can be employed to detect circulating tumor cells(CTCs). The virus that expresses the β-glucuronidase (or other reporter)is administered, and then body fluids, such as blood and serum andplasma, that can contain CTCs are sampled. A substrate for the encodedenzyme is added to the sample and detection of the product of enzyme andsubstrate indicates the presence of circulating tumor cells. The cellscan be quantified, and their present and numbers measured over time toassess therapeutic effectiveness of the any therapy.

Another exemplary enzymatic reporter protein is β-galactosidase,including any form thereof, prokaryotic or eukaryotic or modified forms.Exemplary substrates therefore include, but are not limited to,4-Methylumbelliferyl β-D-galactopyranoside, Fluoresceinβ-D-galactopyranoside (FDG), 5-(Pentafluorobenzoylamino)-fluoresceinβ-D-galactopyranoside, C2-Fluorescein β-D-galactopyranoside,C12-Fluorescein galactopyranoside, 5-Chloromethylfluoresceinβ-D-galactopyranoside, C12-Resorufin, DDAO and Resorufin and5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, BCIG).

Luciferase is another exemplary enzyme. Substrates therefore typicallyare luciferins. Luciferase can be those from any bacterial or plantspecies and their corresponding substrate, such as click beetleluciferin, firefly luciferin, latia luciferin, bacterial luciferin,Renilla luciferin, coelenterazine luciferin, dinoflagellate luciferinand cypridina luciferin.

Body fluid samples for practicing the methods, include, but are notlimited to, a body fluid that is selected from among blood, plasma,serum, lymph, ascetic fluid, cystic fluid, urine, nipple exudates,sweat, tears, saliva, mouth gargle, peritoneal fluid, cerebrospinalfluid (csf), synovial fluid, aqueous humour, vitreous humour, amnioticfluid, bile, cerumen (earwax), Cowper's fluid (pre-ejaculatory fluid),Chyle, Chyme, female ejaculate, interstitial fluid, lymph fluid, menses,breast milk, mucus, snot, phlegm, pleural fluid, pus, sebum, semen,vaginal lubrication, and feces. Exemplary of convenient samples areblood, plasma, serum and urine, such as blood, or such as urine.

The sample can be collected once or periodically or intermittently. Itcan be collected before treatment for comparison with a sample orsamples subsequent to treatment. Exemplary time periods include, forexample, collection of sample between or between about 12 hours to 1month, 12 hours to 2 weeks, 12 hours to 7 days, 1 day to 7 days, 1 dayto 5 days, 1 day to 3 days, 1 day to 2 days, 1 week to 4 weeks, 1 weekto 3 weeks, 1 week to 2 weeks after treatment with the vector orbiological therapy, or is collected on or on about 12 hours, 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days 21 days, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeksafter treatment with the vector or biological therapy or between orbetween about 12 hours to 1 month, 12 hours to 2 weeks, 12 hours to 7days, 1 day to 7 days, 1 day to 5 days, 1 day to 3 days, 1 day to 2days, 1 week to 4 weeks, 1 week to 3 weeks, 1 week to 2 weeks aftertreatment with the vector or biological therapy, or is collected on oron about 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 4 weeks, 5weeks, 6 weeks, 7 weeks or 8 weeks after treatment with the vector orbiological therapy.

The amount of sample or treated sample or purified protein from a sampleand substrate can be empirically determined or is known. Exemplaryratios of substrate to sample include, but are not limited to, from orfrom about 1:1,000,000 to 1:100,000; 1:1,000,000 to 1:10,000;1:1,000,000 to 1:1,000; 1:500,000 to 1:100,000; 1:500,000 to 1:50,000;1:500,000 to 1:10,000; 1:100,000 to 10,000; 1:100,000 to 1:1,000;1:100,000 to 1:500; 1:50,000 to 1:10,000; 1:50,000 to 1:1,000; 1:50,000to 1:500; 1:10,000 to 1:1,000; 1:10,000 to 1:500; 1:10,000 to 1:100;1:10,000 to 1:1; 1:1000 to 1:500; 1:1000 to 1:100; 1:1000 to 1:1; 1:500to 1:100; 1:500 to 1:1, weight/volume (w/v); or is at least or at leastabout or is 1:1,000,000, 1:500,000, 1:250,000, 1:100,000, 1:75,000,1:50,000, 1:25,000, 1:10,000, 1:5,000, 1:2,500, 1:1,000, 1:900, 1:800,1:700, 1:600, 1:500, 1:400, 1:300, 1:200, 1:100, 1:50, 1:45, 1:40, 1:35,1:30, 1:25, 1:20, 1:15, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1 or less, weight tovolume (w/v).

Substrate can be added to a sample in any suitable amount, such as, forexample, at an amount less than 1 pg, or between or between about 1 pgto 1 mg, 1 pg to 1 μg, 1 pg to 1 ng, 1 pg to 500 pg, 50 pg to 1 mg, 50pg to 1 μg, 50 pg to 1 ng, 50 pg to 500 pg, 250 pg to 1 μg, 250 pg to 1ng, 250 pg to 750 pg, 500 pg to 1 mg, 500 pg to 1 μg, 500 pg to 1 ng, 1ng to 1 mg, 1 ng to 500 μg, 1 ng to 1 μg, 1 ng to 500 ng, 250 ng to 1mg, 250 ng to 500 μg, 250 ng to 250 μg, 250 ng to 1 μg, 250 ng to 750ng, 500 ng to 1 mg, 500 ng to 500 μg, 500 ng to 1 μg, 1 μg to 1 mg, 1 μgto 500 μg, 1 μg to 250 μg, 1 μg to 100 μg, 1 μg to 50 μg, 1 μg to 10 μg,10 μg to 1 mg, 10 μg to 500 μg, 10 μg to 250 μg, 10 μg to 100 μg, 10 μgto 50 μg, 25 μg to 500 μg, 25 μg to 250 μg, 25 μg to 100 μg, 50 μg to 1mg, 50 μg to 500 μg, 50 μg to 250 μg, 50 μg to 100 μg, 100 μg to 1 mg,100 μg to 500 μg, 100 μg to 300 μg, 300 μg to 700 μg, 300 μg to 500 μg,500 μg to 1 mg; or is about or at least about or is 1 pg, 5 pg, 10 pg,20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350pg, 400 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850pg, 900 pg, 950 pg, 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 150ng, 200 ng, 250 ng, 300 ng, 350 ng, 400 ng, 450 ng, 500 ng, 550 ng, 600ng, 650 ng, 700 ng, 750 ng, 800 ng, 850 ng, 900 ng, 950 ng, 1 μg, 1.25μg, 1.5 μg, 1.75 μg, 2 μg, 2.25 μg, 2.5 μg, 2.75 μg, 3 μg, 3.25 μg, 3.5μg, 3.75 μg, 4 μg, 4.25 μg, 4.5 μg, 4.75 μg, 5 μg, 5.5 μg, 6 μg, 6.5 μg,7 μg, 7.5 μg, 8 μg, 8.5 μg, 9 μg, 9.5 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg,250 μg, 500 μg or 1 mg. Contacting a sample with a substrate can beeffected for a suitable time, including incubation for less than aminute, from about 1 minute to 2 hours, 1 minute to 1 hour, 1 minute to30 minutes, 1 minute to 15 minutes, 15 minutes to 2 hours, 15 minutes to1 hour, 15 minutes to 45 minutes, 15 minutes to 30 minutes, 30 minutesto 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30 minutes to1 hour, 1 hour to 24 hours, 1 hour to 18 hours, 1 hour to 12 hours, 1hour to 6 hours, 1 hour to 3 hours, 1 hour to 2 hours, 6 hours to 24hours, 6 hours to 18 hours, 6 hours to 12 hours, 12 hours to 24 hours,12 hours to 18 hours, or is at least or is about or is 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 12 hours, 18 hours or 24 hours prior to detection.

The methods herein can be employed for detecting a bacterial infectionby obtaining a sample from a subject and adding a detectable substratefor beta-glucuronidase to a sample from a subject suspected of having aninfection; and detecting beta-glucuronidase activity in the sample.Samples include urine, blood and serum. Infections include, for example,among sepsis, and a urinary tract infection. The substrates are anysuitable substrates for such enzyme, including those listed above, suchas, but not limited to fluorescein di-β-D-glucuronide (FDGlcU) and4-methylumbelliferyl-β-D-glucuronide (4-MUG). Bacterial infectionsinclude, but are not limited to, those caused by Escherichia, Shigellaand Salmonella, and any microorganism that expresses abeta-galactosidase. The methods can further include administering anantibiotic effective against such bacteria.

Similarly, method for detecting viral infection of a cell are provided.The cells include cells that occur in vivo and also in in vitrocultures. A sample from a tissue or body fluid of a host infected withthe virus or from culture medium in which the cells are cultured isobtained that a product encoded by the virus, particularly a vacciniavirus, including any described herein, and any that encode reporters,including a beta-glucuronidase, as described herein, is detected. Asnoted the methods can be used to detect a tumor, particularly byadministering a vaccinia virus, including any describe herein or knownto those of skill in the art, obtaining a body fluid sample, anddetecting beta-glucuronidase activity in the sample, wherein detectionof activity indicates the presence of tumor cells in the subject.

Also provided herein is a method for monitoring oncolytic viral therapythat involves obtaining a sample from a subject treated with thetherapy, wherein the subject had been treated with a virus that encodesbeta-glucuronidase; and the sample is a body fluid or tissue sample andis not a tumor sample; and detecting beta-glucuronidase activity in thesample, wherein activity in the sample indicates that the therapeuticvirus has colonized or is replicating in a tumor. In some examples, thenucleic acid encoding the beta-glucuronidase is not operatively linkedto nucleic acid encoding a signal sequence for secretion. In otherexamples, nucleic acid encoding the beta-glucuronidase is operativelylinked to nucleic acid encoding a signal sequence for secretion. In theprovided method, sampling can be repeated periodically or intermittentlyfollowing treatment(s).

The oncolytic virus used in the provided method can be selected fromamong an oncolytic poxvirus, adenovirus, reovirus, herpes virus,adeno-associated virus, lentivirus, retrovirus, rhabdovirus,papillomavirus, vesicular stomatitis virus, measles virus, Newcastledisease virus, picornavirus, sindbis virus, papillomavirus, parvovirus,coxsackievirus, influenza virus, mumps virus, poliovirus and semlikiforest virus. In some examples, the virus is a poxvirus that is selectedfrom among an orthopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus,leporipoxvirus, suipoxvirus, molluscipoxvirus, yatapoxvirus,entomopoxvirus A, entomopoxvirus B and entomopoxvirus C. In an exemplaryexample, the virus is a poxvirus. The poxvirus used in the method can bea vaccinia virus that is an LIVP virus, clonal variant of an LIVP virus.In other examples, the vaccinia virus is a Lister strain virus that isLIVP or a clonal variant of LIVP. In an exemplary example of the method,the virus is GLV-1h68 or a derivative or modified form thereof. Infurther examples of the method, the virus has a sequence of nucleotidesset forth in any of SEQ ID NOS:82-83 and 85-91, or a sequence ofnucleotides that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8% or 99.9% sequence identity to a sequence ofnucleotides set forth in any of SEQ ID NOS: 82-83 and 85-91. In someexamples, the virus is selected from among GLV-1h22, GLV-1i69, GLV-1h70,GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h80, GLV-1h81, GLV-1h82,GLV-1h83, GLV-1h84, GLV-1h85, GLV-1h86, GLV-1j87, GLV-1j88, GLV-1j89,GLV-1h90, GLV-1h91, GLV-1h92, GLV-1h96, GLV-1h97, GLV-1h98, GLV-1h99,GLV-1h100, GLV-1h101, GLV-1h104, GLV-1h105, GLV-1h106, GLV-1h107,GLV-1h108, GLV-1h109, GLV-1h139, GLV-1h146, GLV-1h150, GLV-1h151,GLV-1h152 and GLV-1h153.

In the provided method, the biological therapeutic can be administeredsystemically. In other examples, administration of the biologicaltherapeutic is effected topically, locally, enterally or parenterally,for example, administration is effected orally, epicutaneously,intranasally, by gastric feeding tube, duodenal feeding tube, orgastrostomy, rectally, intravenously, intraarterially, intramuscularly,intracardiacly, subcutaneously, by intraosseous infusion (into the bonemarrow), intradermally, intraperitoneally, intrapleurally,transdermally, transmucosally, epiduralally, intrathecally,intraventricularly or intratumorally.

In some examples of the provided method, the amount of virusadministered is 1×10⁵ or about 1×10⁵ plaque forming units (PFU), 5×10⁵or about 5×10⁵ PFU, 1×10⁶ or about 1×10⁶ PFU, 5×10⁶ or about 5×10⁶ PFU,1×10⁷ or about 1×10⁷ PFU, 5×10⁷ or about 5×10⁷ PFU, 1×10⁸ or about 1×10⁸PFU, 5×10⁸ or about 5×10⁸ PFU, 1×10⁹ or about 1×10⁹ PFU, 5×10⁹ or about5×10⁹ PFU, 1×10¹⁰ or about 1×10¹⁰ PFU or 5×10¹⁰ or about 5×10¹⁰ PFU astotal single dosage for an average human of 75 kg or adjusted for theweight and size and species of the subject. One of skill in the art candetermine suitable dosage. Suitable dosages for a virus can bedetermined empirically.

In the methods herein, the β-glucuronidase can be a human or bacterialβ-glucuronidase. In an exemplary embodiment, the β-glucuronidase has asequence of amino acids set forth in SEQ ID NO:121 or an active portionthereof or a sequence having at least 85% sequence identity to asequence of amino acids set forth in SEQ ID NO:121. In yet anotherexemplary embodiment, the β-glucuronidase has a sequence of amino acidsset forth in SEQ ID NO:4 or an active portion thereof or a sequencehaving at least 85% sequence identity to a sequence of amino acids setforth in SEQ ID NO:4. Other β-glucuronidase enzymes include any having asequence of amino acids set forth in any of SEQ ID NOS: 4, 114-121, 128,130, 132, 134, 136, 138, 140, 142, 144 and 146, or an active portionthereof or a sequence having at least 85% sequence identity to asequence of amino acids set forth in any of SEQ ID NOS: 4, 114-121, 128,130, 132, 134, 136, 138, 140, 142, 144 and 146.

In some examples of the provided method, the substrate is selected fromamong fluorescein di-β-D-glucuronide (FDGlcU),4-methylumbelliferyl-β-D-glucuronide (4-MUG), carboxyumbelliferylβ-D-glucuronide (CUGlcU), 5-(Pentafluorobenzoylamino)fluoresceindi-β-D-glucuronide (PFB-FDGlcU), C₁₂-Fluorescein β-D-Glucuronidase,5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GlcU or BCIG),p-nitrophenyl-β-D-glucuronide, Red-β-D-GlcU,CHA (Magenta-b-D-GlcA;5-bromo-6-chloro-3-indolyl-b-D-glucuronide, cyclohexylammonium salt),Rose-β-D-GlcU,CHA (Salmon-β-D-GlcUA;5-bromo-6-chloro-3-indolyl-β-D-glucu-ronide, cyclohexylammonium salt),phenyl-β-D-glucuronide, and suitable salts thereof. In an exemplaryembodiment, the substrate is selected from among fluoresceindi-β-D-glucuronide (FDGlcU) and 4-methylumbelliferyl-β-D-glucuronide(4-MUG).

Body fluid samples for practicing the methods, include, but are notlimited to, blood, plasma, serum, lymph, ascetic fluid, cystic fluid,urine, nipple exudates, sweat, tears, saliva, mouth gargle, peritonealfluid, cerebrospinal fluid (csf), synovial fluid, aqueous humour,vitreous humour, amniotic fluid, bile, cerumen (earwax), Cowper's fluid(pre-ejaculatory fluid), Chyle, Chyme, female ejaculate, interstitialfluid, lymph fluid, menses, breast milk, mucus, snot, phlegm, pleuralfluid, pus, sebum, semen, vaginal lubrication, and feces. In someexamples, the sample is blood, plasma, serum or urine. In an exemplaryembodiment, the sample is serum.

Subjects include any subject, including but not limited to any animal.The animals include humans, veterinary animals, pets, farm animals,animal models. Included are human, cats, dogs, frogs, ferrets, gorillas,chimpanzees, gorillas, birds, lions, tigers, cows, rodents, goats, pigs,chickens, horses, zebras, dolphins and whales.

The sample can be collected once or periodically or intermittently. Itcan be collected before treatment for comparison with a sample orsamples subsequent to treatment. Exemplary time periods include, forexample, collection of sample between or between about 12 hours to 1month, 12 hours to 2 weeks, 12 hours to 7 days, 1 day to 7 days, 1 dayto 5 days, 1 day to 3 days, 1 day to 2 days, 1 week to 4 weeks, 1 weekto 3 weeks, 1 week to 2 weeks after treatment with the vector orbiological therapy, or is collected on or on about 12 hours, 1 day, 2days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeksafter treatment with the virus. In other examples, one or more samplesare collected between or between about 12 hours to 1 month, 12 hours to2 weeks, 12 hours to 7 days, 1 day to 7 days, 1 day to 5 days, 1 day to3 days, 1 day to 2 days, 1 week to 4 weeks, 1 week to 3 weeks, 1 week to2 weeks after treatment with the vector or biological therapy, or iscollected on or on about 12 hours, 1 day, 2 days, 3 days, 4 days, 5days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21days, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks after treatment withthe virus.

The amount of sample or treated sample or purified protein from a sampleand substrate can be empirically determined or is known. Exemplaryratios of substrate to sample include, but are not limited to, from orfrom about 1:1,000,000 to 1:100,000; 1:1,000,000 to 1:10,000;1:1,000,000 to 1:1,000; 1:500,000 to 1:100,000; 1:500,000 to 1:50,000;1:500,000 to 1:10,000; 1:100,000 to 10,000; 1:100,000 to 1:1,000;1:100,000 to 1:500; 1:50,000 to 1:10,000; 1:50,000 to 1:1,000; 1:50,000to 1:500; 1:10,000 to 1:1,000; 1:10,000 to 1:500; 1:10,000 to 1:100;1:10,000 to 1:1; 1:1000 to 1:500; 1:1000 to 1:100; 1:1000 to 1:1; 1:500to 1:100; 1:500 to 1:1, weight/volume (w/v); or is at least or at leastabout or is 1:1,000,000, 1:500,000, 1:250,000, 1:100,000, 1:75,000,1:50,000, 1:25,000, 1:10,000, 1:5,000, 1:2,500, 1:1,000, 1:900, 1:800,1:700, 1:600, 1:500, 1:400, 1:300, 1:200, 1:100, 1:50, 1:45, 1:40, 1:35,1:30, 1:25, 1:20, 1:15, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1 or less, weight tovolume (w/v). Substrate can be added to a sample in any suitable amount,such as, for example, at an amount less than 1 pg, or between or betweenabout 1 pg to 1 mg, 1 pg to 1 μg, 1 pg to 1 ng, 1 pg to 500 pg, 50 pg to1 mg, 50 pg to 1 μg, 50 pg to 1 ng, 50 pg to 500 pg, 250 pg to 1 μg, 250pg to 1 ng, 250 pg to 750 pg, 500 pg to 1 mg, 500 pg to 1 μg, 500 pg to1 ng, 1 ng to 1 mg, 1 ng to 500 μg, 1 ng to 1 μg, 1 ng to 500 ng, 250 ngto 1 mg, 250 ng to 500 μg, 250 ng to 250 μg, 250 ng to 1 μg, 250 ng to750 ng, 500 ng to 1 mg, 500 ng to 500 μg, 500 ng to 1 μg, 1 μg to 1 mg,1 μg to 500 μg, 1 μg to 250 μg, 1 μg to 100 μg, 1 μg to 50 μg, 1 μg to10 μg, 10 μg to 1 mg, 10 μg to 500 μg, 10 μg to 250 μg, 10 μg to 100 μg,10 μg to 50 μg, 25 μg to 500 μg, 25 μg to 250 μg, 25 μg to 100 μg, 50 μgto 1 mg, 50 μg to 500 μg, 50 μg to 250 μg, 50 μg to 100 μg, 100 μg to 1mg, 100 μg to 500 μg, 100 μg to 300 μg, 300 μg to 700 μg, 300 μg to 500μg, 500 μg to 1 mg; or is about or at least about or is 1 pg, 5 pg, 10pg, 20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg,350 pg, 400 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg,850 pg, 900 pg, 950 pg, 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng,150 ng, 200 ng, 250 ng, 300 ng, 350 ng, 400 ng, 450 ng, 500 ng, 550 ng,600 ng, 650 ng, 700 ng, 750 ng, 800 ng, 850 ng, 900 ng, 950 ng, 1 μg,1.25 μg, 1.5 μg, 1.75 μg, 2 μg, 2.25 μg, 2.5 μg, 2.75 μg, 3 μg, 3.25 μg,3.5 μg, 3.75 μg, 4 μg, 4.25 μg, 4.5 μg, 4.75 μg, 5 μg, 5.5 μg, 6 μg, 6.5μg, 7 μg, 7.5 μg, 8 μg, 8.5 μg, 9 μg, 9.5 μg, 10 μg, 11 μg, 12 μg, 13μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 25 μg, 30 μg, 35μg, 40 μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200μg, 250 μg, 500 μg or 1 mg. Contacting a sample with a substrate can beeffected for a suitable time, including incubation for less than aminute, from or from about 1 minute to 2 hours, 1 minute to 1 hour, 1minute to 30 minutes, 1 minute to 15 minutes, 15 minutes to 2 hours, 15minutes to 1 hour, 15 minutes to 45 minutes, 15 minutes to 30 minutes,30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30minutes to 1 hour, 1 hour to 24 hours, 1 hour to 18 hours, 1 hour to 12hours, 1 hour to 6 hours, 1 hour to 3 hours, 1 hour to 2 hours, 6 hoursto 24 hours, 6 hours to 18 hours, 6 hours to 12 hours, 12 hours to 24hours, 12 hours to 18 hours, or is at least or is about or is 1 minute,2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 12 hours, 18 hours or 24 hours prior to detection. For example,the sample and substrate are incubated for at least or about at least or1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45minutes, 50 minutes, 55 minutes or 1 hour prior to detection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Fluorogenic compound activation in rVACV-colonized tumors. Tumorspecific fluorescence over time post FDGlcU-injection (n=4; average plusstandard deviation is shown).

FIG. 2. Time dependent conversion of FDGlcU in the same mouse injectedwith GLV-1h68. An A549 Tumor-bearing mouse was injected with GLV-1h68 10days before FDGlcU injection was performed. Intraperitoneal (i.p.)injection (lower row pictures) occurred 24 hours before intravenous(i.v.) injection (upper row pictures). This allowed the fluorescencesignal to decline completely before getting the kinetics in the verysame mouse.

FIG. 3. Analysis of urine samples derived from mice before, 30 min and90 min post FDGlcU injection respectively. Three and seven days postrVACV injection urine was sampled before and 90 min post FDGlcUinjection. Average plus standard deviation of fluorescein specificfluorescence of GusA-positive (n=6 for 3 dpi and n=8 for 7 dpi) andGusA-negative (n=4) rVACV colonized tumors. * indicates p<0.05.

FIG. 4. Glucuronidase specific fluorogenic compound activation in serumof tumor bearing mice. FIG. 4A. Tumor bearing mice were mock injected(n=2) or injected with GLV-1h68 (n=6) and rVACV-GusA⁻ (n=6)respectively. Seven days later 5 μl serum was co-incubated for 1 h at37° C. with 4-MUG and FDGlcU respectively and subsequently specificfluorescence was determined. FIG. 4B. Retrospective serum analysis.Serum samples (n=99) from different tumor xenograft models (GI-101A,A549, DU-145, PANC-1, HT-29) were retrospectively tested. Samples werederived from mock (n=33) injected mice or mice treated for differentperiods of time (7 to 53 days) with several GusA-positive (n=53) or-negative (n=13) rVACV. * indicates p<0.03; *** indicates p<0.001.

FIG. 5. Positive correlation between the fluorescence signal intensitiesand increasing glucuronidase concentration, fluorogenic substrateconcentration (left panels, 4-MUG in FIG. 5A., FDGlcU in FIG. 5B. andincubation time (right panels).

FIG. 6. Minimal amount of GLV-1h68 infected cancer cells necessary forpositive detection. A549 cells were infected with GLV-1h68 orcontrol-rVACV (rVACV-GusA^(neg)). One day later, the amount of infectedcells was determined by flow cytometry and the cells were seeded inhalf-log dilutions in 384-well plates with concentrations varying from1.0 to 1000 cells/well and co-incubated with FDGlcU and 4-MUGrespectively. Data represent average plus standard deviation (n=6).

DETAILED DESCRIPTION Outline

A. Definitions

B. Overview of Method

-   -   1. Biological Therapies        -   a. Oncolytic Viral Therapy        -   b. other viruses    -   2. Beta-glucuronidases as reporter proteins    -   3. Method

C. Method for Detecting Replication or Colonization of a BiologicalTherapeutic

-   -   1. Biological Therapeutics        -   a. Viruses            -   i. Poxviruses                -   (1) Vaccinia Viruses                -   (2) Modified Vaccinia Viruses                -   (3) Exemplary Modified Vaccinia Viruses            -   ii. Other Cytoplasmic Viruses            -   iii. Adenovirus, Herpes, Retroviruses        -   b. Bacteria            -   i. E. coli strain Nissle 1917            -   ii. Other bacteria        -   c. Other Therapies            -   i. Gene Therapy            -   ii. Cell Therapy            -   iii. Immunotherapy    -   2. Reporters Proteins        -   a. Reporter Enzymes            -   i. β-glucuronidases            -   ii. β-galactosidases            -   iii. Luciferases            -   iv. Chloramphenicol Acetyltransferases            -   v. Alkaline phosphatases        -   b. Reporter Enzyme Substrates            -   i. β-glucuronidase substrates            -   ii. β-galactosidase substrates            -   iii. Luciferase substrates            -   iv. Chloramphenicol Acetyltransferase substrates            -   v. Alkaline phosphatase substrates    -   3. Sample    -   4. Addition of Reporter Protein Substrates    -   5. Incubation of the Sample and the Reporter Protein Substrate    -   6. Detection of Activated Substrate or Signal

D. Methods of Making Biological Therapeutics encoding an ReporterProtein

-   -   1. Vectors    -   2. Viruses        -   a. Genetic Modifications        -   b. Control of heterologous gene expression    -   3. Bacteria

E. Methods for detecting and monitoring therapy

F. Examples

A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. All patents, patent applications,published applications and publications, Genbank sequences, databases,websites and other published materials referred to throughout the entiredisclosure herein, unless noted otherwise, are incorporated by referencein their entirety. In the event that there are a plurality ofdefinitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information can be foundby searching the internet. Reference thereto evidences the availabilityand public dissemination of such information.

As used herein, a sample from a locus other than a target of thebiological therapy, means that the sample is taken from a cell otherthan from the target of the biological therapy. For example, if thetherapy treats tumors, then the sample is not a sample of the tumortissue, but is obtained from another locus. Typically the other locus isa body fluid, but it can include tissue samples. The non-target sample,can include tumor cells, but, if it does, the sample or results aretreated or tested so that tumor or target tissue or cells or biologicaltherapeutic are not present or the results are normalized to eliminateany contribution from tumor or target cells or tissue or biologicaltherapeutic. For purposes herein, the assessed material is distinct fromthe tumor or target cells or tissue or biological therapeutic andmeasures or detects shed proteins that are encoded by the biologicaltherapeutic. For gene therapy and other therapies in which a particularproduct is introduced, the tested product is not the gene therapyproduct, but is another product encoded by the biological therapeutic.

As used herein, shed proteins means that the encoded proteins are notactively secreted, but are produced by the biological therapeutic andappear in biological tissues and fluids because of replication andcolonization by the biological therapeutic. In particular, inembodiments in which the biological therapeutic is an oncolytic virus,such as a vaccinia virus, the encoded protein is shed by virtue of thefact that the virus replicates in tumor cells and lyses such cells,which releases the proteins.

As used herein, a target of the therapeutic refers to the intended locusof treatment, e.g., the target locus. If the therapeutic is an oncolyticvirus, the target is/are tumor cells or other immunoprivileged cells. Ifthe therapy is gene therapy to provide a gene product that issystemically expressed, then the target is systemic expression, and asample from a locus other than the target means that a sample is treatedor detection is effected or normalized to eliminate any contributionfrom the introduced gene therapy biological therapeutic or an encodedproduct other than gene therapy product is detected.

As used herein, a reporter gene refers to nucleic acid contained withina biological therapeutic that encodes a reporter protein that is to bedetected. The reporter gene does not encode a therapeutic productencoded by the biological therapeutic, but encodes some other endogenousor heterologous product that is to be detected, either directly orindirectly. Typically, the reporter gene is heterologous to thebiological therapeutic and encodes a detectable protein or encodes aprotein that induces or produces a detectable signal. In embodiments inwhich the encoded reporter protein is shed, the reporter gene is notoperatively linked to nucleic acid that directs secretion of theproduct. In embodiments, such as embodiments in which the gene encodes aglucuronidase, it can be secreted.

As used herein, a reporter protein is a protein encoded by a reportergene. The reporter protein is not a therapeutic protein encoded by thebiological therapeutic, but is some other endogenous or heterologousprotein that is to be detected, either directly or indirectly.Typically, the reporter protein is heterologous to the biologicaltherapeutic and is detectable or is a protein that induces or produces adetectable signal. A reporter protein can be shed, that is, the reporterprotein is not secreted by the biological therapeutic. In embodimentswhere the reporter protein is a beta-glucuronidase, the reporter proteincan be shed or secreted by the biological therapeutic. A reporter enzymeor enzymatic reporter protein refers to a reporter protein that is anenzyme. Detection of the reporter protein, including reporter enzyme, orsignal induced by a reporter protein is indicative of gene expression ofa gene contained with the biological therapeutic, such as the vector orvirus.

As used herein, a sample is a body fluid or tissue sample from a subjectthat has been or will be treated with a biological therapeutic. Thesample is tested to detect a product, such as a reporter protein, thathas been expressed and shed or, in some instances, such as where thereporter is beta-glucuronidase and the biological therapeutic is avaccinia virus, secreted or shed, by the biological therapeutic. Theresults are compared to an appropriate control and/or corrected, so thatonly the contribution of the particular reporter protein is determined.These results assess the colonization or replication of the biologicaltherapeutic in a target cell or cells or target tissue(s). Examples ofbody fluids include urine, blood, plasma, serum, saliva, semen, stool,sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.

As used herein, biological therapy refers to any therapy that involvesadministration of a biological agent or biological therapeutic, such asa virus, a viral vector and a cell, including stem cells and cells usedfor adoptive immunotherapy protocols, for purposes of treatment and/ordiagnostics. Hence included are therapies in which mammalian viruses forgene therapy are administered, therapies in which eukaryotic orprokaryotic organisms, such as cells, viruses and bacteria, areadministered for treatment or diagnosis, and oncolytic viral therapies,particularly those in which viruses that accumulate or that are modifiedto accumulate in immunoprivileged cells, such as tumor cells andinflamed tissues, are administered. The administered cells and virusesand bacteria can be modified by inclusion of heterologous nucleic acidthat encodes therapeutic products and/or reporters. The methods hereinassess expression of a product or a signal produced by a product encodedby the biological therapy in a sample, typically a body fluid sample,obtained from a subject to whom such virus, cell and/or bacterium isadministered. The target of the therapy, such as a tumor tissue or othertissue, is different from the locus from which the sample is obtained.

As used herein, replication of a biological therapeutic in a host orsubject refers to replication of a virus, bacteria, or a genome thereof,in a host or subject to whom such therapeutic is administered. Fortherapies such as oncolytic viral therapies and gene therapies requiringsustained expression of a product or cell therapies, treatment requiresreplication of the genome of the therapeutic. For the detection methodsherein, replication is indicative that the therapy is initiated.Colonization generally refers to establishment of a biologicaltherapeutic in a host. Biological therapies such as cell therapies inwhich cells are transplanted requires that an organ or tissue iscolonized by the administered cells to produce a product. Colonizationand replication are not mutually exclusive.

As used herein, circulating tumor cell or CTC refers to a tumor cellderived from a primary cancer site that has detached from the primarytumor mass. CTCs includes cancer cells, malignant tumor cells, benigntumor cells and cancer stem cells. CTCs include any cancer cell orcluster of cancer cells that is found in a sample obtained from asubject. CTCs are often epithelial cells shed from solid tumors. CTCsalso can be mesothelial cells from carcinomas or melanocytes frommelanomas. A CTC is typically a cell originating from a primary tumor,but also can be a cell shed from a metastatic tumor (e.g., a secondaryor tertiary tumor). As used herein, the term “CTC” is intended toencompass any tumor cell that has detached from a tumor. Thus, as usedherein, a CTC encompasses tumor cells found in circulation, such as inthe blood or lymph, or in other fluid samples, such as, but not limitedto, pleural fluid, peritoneal fluid, central spinal fluid, abdominalfluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites,urine, saliva, bronchial lavage, bile, sweat, tears, ear flow, sputum,semen, vaginal flow, milk, amniotic fluid, and secretions ofrespiratory, intestinal or genitourinary tract. The term CTC as usedherein also includes disseminated tumor cells (DTCs) found in the bonemarrow.

As used herein the term “vaccinia virus” or “VACV” denotes a large,complex, enveloped virus belonging to the poxvirus family. It has alinear, double-stranded DNA genome approximately 190 kbp in length, andwhich encodes approximately 200 proteins. Vaccinia virus strainsinclude, but are not limited to, strains of, derived from, or modifiedforms of Western Reserve (WR), Copenhagen, Tashkent, Tian Tan, Lister,Wyeth, IHD-J, and IHD-W, Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8,LC16MO, LIVP, WR 65-16, Connaught, New York City Board of Healthvaccinia virus strains.

As used herein, Lister Strain of the Institute of Viral Preparations(LIVP) or LIVP virus strain refers to a virus strain that is theattenuated Lister strain (ATCC Catalog No. VR-1549) that was produced byadaption to calf skin at the Institute of Viral Preparations, Moscow,Russia (Al'tshtein et al. (1985) Dokl. Akad. Nauk USSR 285:696-699). TheLIVP strain can be obtained, for example, from the Institute of ViralPreparations, Moscow, Russia (see. e.g., Kutinova et al. (1995) Vaccine13:487-493); the Microorganism Collection of FSRI SRC VB Vector (Kozlovaet al. (2010) Environ. Sci. Technol. 44:5121-5126); or can be obtainedfrom the Moscow Ivanovsky Institute of Virology (C0355 K0602; Agranovskiet al. (2006) Atmospheric Environment 40:3924-3929). It also is wellknown to those of skill in the art; it was the vaccine strain used forvaccination in the USSR and throughout Asia and India. The strain now isused by researchers and is well known (see e.g., Altshteyn et al. (1985)Dokl. Akad. Nauk USSR 285:696-699; Kutinova et al. (1994) Arch. Virol.134:1-9; Kutinova et al. (1995) Vaccine 13:487-493; Shchelkunov et al.(1993) Virus Research 28:273-283; Sroller et al. (1998) ArchivesVirology 143:1311-1320; Zinoviev et al., (1994) Gene 147:209-214; andChkheidze et al. (1993) FEBS 336:340-342). Among the LIVP strains is onethat contains a genome having a sequence of nucleotides set forth in SEQID NO:91, or a sequence that is at least or at least about 99% identicalto the sequence of nucleotides set forth in SEQ ID NO:91.

As used herein, an LIVP clonal strain, LIVP clonal variant or LIVPclonal isolate refers to a virus that is derived from the LIVP virusstrain by plaque isolation, or other method in which a single clone ispropagated, and that has a genome that is homogenous in sequence. Hence,an LIVP clonal strain includes a virus whose genome is present in avirus preparation propagated from LIVP. An LIVP clonal strain does notinclude a recombinant LIVP virus that is genetically engineered byrecombinant means using recombinant DNA methods to introduceheterologous nucleic acid. In particular, an LIVP clonal strain has agenome that does not contain heterologous nucleic acid that contains anopen reading frame encoding a heterologous protein. For example, an LIVPclonal strain has a genome that does not contain non-viral heterologousnucleic acid that contains an open reading frame encoding a non-viralheterologous protein. As described herein, however, it is understoodthat any of the LIVP clonal strains provided herein can be modified inits genome by recombinant means to generate a recombinant virus. Forexample, an LIVP clonal strain can be modified to generate a recombinantLIVP virus that contains insertion of nucleotides that contain an openreading frame encoding a heterologous protein. Among the LIVP clonalisolates are those having a genome having a sequence of amino acids setforth in SEQ ID NOS:82-83 and 85-89, or sequences that are at least orat least about 99% identical to the sequences of nucleotides set forthin SEQ ID NOS:82-83 and 85-89.

As used herein, a modified LIVP virus strain refers to an LIVP virusthat has a genome that is not contained in LIVP, but is a virus that isproduced by modification of a genome of a strain derived from LIVP.Typically, the genome of the virus is modified by substitution(replacement), insertion (addition) or deletion (truncation) ofnucleotides. Modifications can be made using any method known to one ofskill in the art such as genetic engineering and recombinant DNAmethods. Hence, a modified virus is a virus that is altered in itsgenome compared to the genome of a parental virus. Exemplary modifiedviruses have one or more heterologous nucleic acid sequences insertedinto the genome of the virus. Typically, the heterologous nucleic acidcontains an open reading frame encoding a heterologous protein. Forexample, modified viruses herein can contain one or more heterologousnucleic acid sequences in the form of a gene expression cassette for theexpression of a heterologous gene.

As used herein a “gene expression cassette” or “expression cassette” isa nucleic acid construct, containing nucleic acid elements that arecapable of effecting expression of a gene in hosts that are compatiblewith such sequences. Expression cassettes include at least promoters andoptionally, transcription termination signals. Typically, the expressioncassette includes a nucleic acid to be transcribed operably linked to apromoter. Expression cassettes can contain genes that encode, forexample, a therapeutic gene product, or a detectable protein or aselectable marker gene.

As used herein, LIVP GLV-1h68 is an LIVP virus that contains ruc-gfp (aluciferase and green fluorescent protein fusion gene (see e.g. U.S. Pat.No. 5,976,796), beta-galactosidase (lacZ) and beta-glucuronidase (gusA)reporter genes inserted into the F14.5L, J2R (thymidine kinase) and A56R(hemagglutinin) loci, respectively. The genome of GLV-1h68 has asequence of nucleotides set forth in SEQ ID NO:90, or a sequence ofnucleotides that has at least 99% sequence identity to the sequence ofnucleotides set forth in SEQ ID NO:90.

As used herein, a virus preparation, for example an LIVP viruspreparation, refers to a virus composition obtained by propagation of avirus strain, for example an LIVP virus strain, an LIVP clonal strain ora modified or recombinant virus strain, in vivo or in vitro in a culturesystem. For example, an LIVP virus preparation refers to a viralcomposition obtained by propagation of a virus strain in host cells,typically upon purification from the culture system using standardmethods known in the art. A virus preparation generally is made up of anumber of virus particles or virions. If desired, the number of virusparticles in the sample or preparation can be determined using a plaqueassay to calculate the number of plaque forming units per sample unitvolume (pfu/mL), assuming that each plaque formed is representative ofone infective virus particle. Each virus particle or virion in apreparation can have the same genomic sequence compared to other virusparticles (i.e. the preparation is homogenous in sequence) or can havedifferent genomic sequences (i.e. the preparation is heterogenous insequence). It is understood to those of skill in the art that, in theabsence of clonal isolation, heterogeneity or diversity in the genome ofa virus can occur as the virus reproduces, such as by homologousrecombination events that occur in the natural selection processes ofvirus strains (Plotkin & Orenstein (eds) “Recombinant Vaccinia VirusVaccines” in Vaccines, 3^(rd) edition (1999)).

As used herein, a virus mixture is a virus preparation that contains anumber of virus particles that differ in their genomic sequences. Thevirus mixture can be obtained by infecting a culture system, for examplehost cells, with two or more different virus strains, or one virusstrain and genomic DNA or cloned DNA, followed by propagation andpurification of the resulting virus. For purposes herein, an LIVP viruspreparation can include a virus mixture obtained by propagation of cellsinfected with an LIVP strain and another virus, genomic DNA or clonedDNA, followed by isolation of a virus preparation from the culture,where the preparation contains progeny viruses produced by theinfection. For example, the other virus strain can be poxvirus, such asavipox virus, myxoma virus or other vaccinia virus; a herpesvirus suchas herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus(EBV), hepadnaviruses (e.g., hepatitis B virus), polyoma viruses,papillomaviruses, adenoviruses and adeno-associated viruses; andsingle-stranded DNA viruses, such as parvoviruses. The other virus canbe an attenuated virus, oncolytic virus or other virus with knownanti-tumor activity and/or moderate to mild toxicity.

As used herein, “virus” refers to any of a large group of infectiousentities that cannot grow or replicate without a host cell. Virusestypically contain a protein coat surrounding an RNA or DNA core ofgenetic material, but no semipermeable membrane, and are capable ofgrowth and multiplication only in living cells. Viruses include, but arenot limited to, poxviruses, herpesviruses, adenoviruses,adeno-associated viruses, lentiviruses, retroviruses, rhabdoviruses,papillomaviruses, vesicular stomatitis virus, measles virus, Newcastledisease virus, picornavirus, sindbis virus, papillomavirus, parvovirus,reovirus, coxsackievirus, influenza virus, mumps virus, poliovirus, andsemliki forest virus.

As used herein, oncolytic viruses refer to viruses that replicateselectively in tumor cells in tumorous subjects. Some oncolytic virusescan kill a tumor cell following infection of the tumor cell. Forexample, an oncolytic virus can cause death of the tumor cell by lysingthe tumor cell or inducing cell death of the tumor cell.

As used herein, an “attenuated LIVP virus” with reference to LIVP refersto a virus that exhibits reduced or less virulence, toxicity orpathogenicity compared to LIVP.

As used herein, to attenuate toxicity of a bacterium means to reduce oreliminate deleterious or toxic effects to a host upon administration ofthe bacterium compared to an un-attenuated bacterium. As used herein, abacterium with low toxicity, means that upon administration, thebacterium does not accumulate in organs and tissues in the host to anextent that results in damage or harm to organs, or that impactssurvival of the host to a greater extent than the disease being treateddoes.

As used herein, an attenuated, attenuated pathogenic or non-pathogenicbacterium refers to a bacterium that exhibits reduced or less virulence,toxicity or pathogenicity compared to a non-attenuated or pathogenicbacterium.

As used herein, accumulation of bacteria in a targeted tissue refers tothe distribution of the bacteria throughout the organism after a timeperiod long enough for the microbes to infect the host's organs ortissues. As one skilled in the art will recognize, the time period forinfection of a microbe will vary depending on the microbe, the targetedorgan(s) or tissue(s), the immunocompetence of the host, and dosage.Generally, accumulation can be determined at time point from about lessthan 1 day, about 1 day to about 1 week, about 1 week to about 2, 3 or 4weeks, about 1 month to about 2, 3, 4, 5, 6 months or longer afterinfection with the microbes. For purposes herein, the bacteriapreferentially accumulate in the target tissue, such as a tumor, or siteof cellular proliferation, but are cleared from other tissues and organsin the host to the extent that toxicity of the bacteria is mild ortolerable and at most not fatal. As used herein, preferentialaccumulation refers to accumulation of bacteria at a first location at ahigher level than accumulation at a second location. Thus, bacteria thatpreferentially accumulates in immunoprivileged tissue such as tumorrelative to normal tissues or organs refers to bacteria that accumulatein immunoprivileged tissue, such as tumor, at a higher level(concentration) than the bacteria accumulate in normal tissues ororgans.

As used herein, “commensal” when used in reference to an associationbetween two organisms, is a particular association in which one memberof the association benefits from the association while the other memberis essentially unaffected. In a commensal association of organisms, noneof the members of the association is significantly harmed by thepresence of the other member. Two organisms can form a commensalassociation under particular, but not necessarily all, conditions. Insuch cases, as long as an organism is capable of forming a commensalassociation with the other organism under at least one set ofconditions, the organism is considered to be one that can form acommensal association with the other organism.

As used herein, “mutual” when used in reference to an associationbetween two or more organisms, is a particular association which isadvantageous to both members of the association. In a mutual associationof organisms, none of the members of the association is significantlyharmed by the presence of the other member. Two organisms can form amutual association under particular, but not necessarily all,conditions. In such cases, as long as an organism is capable of forminga mutual association with the other organism under at least one set ofconditions, the organism is considered to be one that can form a mutualassociation with the other organism.

As used herein, a probiotic bacterium refers to a bacterium that confersa benefit to a host in which it can occur. The benefit can be, forexample, an overall health benefit to the host, such as preventing,maintaining remission of, preventing recurrence of, reversing orreducing the symptoms or detrimental effects of a disorder or disease ofthe host. Such disorders/diseases include, but are not limited to,infectious diseases, inflammation, diarrhea (e.g., antibiotic-induceddiarrhea, infectious diarrhea and traveler's diarrhea), inflammatorybowel disease, Crohn's disease, pouchitis and colitis. The benefitconferred by a probiotic bacterium can be stabilization of the hostmicrobiota or microecology, for example, by improving the microbialbalance of the indigenous microflora (Kruis W. (2004) Pharmacol. Ther.20 (Suppl 4): 75-78). Probiotic bacteria can exert their effects in anumber of ways. For example, probiotic bacteria can interfere withharmful properties of other pathogenic bacteria that can occur throughthe production of antimicrobial substances by the probiotic bacteria andinterference with bacterial attachment/penetration to/into host cells. Aprobiotic bacterium also can stimulate a host to produce antimicrobialmolecules, alter a host's immune response, stimulate mucosal barrierfunction or alter immunoregulation, such as by decreasingpro-inflammatory molecules and promoting protective molecules (Sartor RB. (2005) Curr. Opin. Gastroenterol. 21(1): 44-50). Exemplary probioticbacteria include, but are not limited to, E. coli strain Nissle 1917(O6:K5:H1; Mutaflor; Ardeypharm GmbH, Germany; Schultz et al. J.Microbiol. Methods 61(3): 389-398 (2005)). E. coli strain Nissle 1917lacks defined virulence factors such as alpha-hemolysin, other toxins,and mannose-resistant hemagglutinating adhesins (Blum et al. Infection.23(4):234-236 (1996)), P-fimbrial adhesins, and the semiroughlipopolysaccharide phenotype and expresses fitness factors such asmicrocins, ferritins, six different iron uptake systems, adhesins, andproteases, which support its survival and successful colonization of thehuman gut (Grozdanov et al. (2004) J Bacteriol. 186(16): 5432-5441). E.coli Nissle 1917 interferes with bacterial invasion of other bacteriacells via a secreted component (Altenhoefer et al. (2004) FEMS Immunol.Med. Microbiol. 40(3): 223-9). E. coli Nissle 1917 can have plasmids(Mutaflor 06:K5:H1, DSM 6601 by Medipharm, Kageröd, Sweden) or noplasmids (i.e. can be cured of plasmids).

As used herein, “toxicity” (also referred to as virulence orpathogenicity herein) with reference to a virus refers to thedeleterious or toxic effects to a host upon administration of the virus.For an oncolytic virus, such as LIVP, the toxicity of a virus isassociated with its accumulation in non-tumorous organs or tissues,which can impact the survival of the host or result in deleterious ortoxic effects. Toxicity can be measured by assessing one or moreparameters indicative of toxicity. These include accumulation innon-tumorous tissues and effects on viability or health of the subjectto whom it has been administered, such as effects on weight.

As used herein, a “parameter indicative of toxicity” refers to aproperty mediated by a virus that is associated with its toxicity,virulence or pathogenicity. Parameters indicative of toxicity generallyare assessed in vivo upon administration to a subject. Exemplaryparameters indicative of toxicity include, but are not limited to,decreased survival of the subject, decreased body weight, fever, rash,allergy, fatigue, abdominal pain, induction of an immune response in thesubject and pock formation. Assays or measures that assess any of theabove parameters or other toxic properties known to one of skill in theart are described herein or are known to one of skill in the art. Hence,a virus that mediates any one or more of the above activities orproperties in a host exhibits some degree of toxicity.

As used herein, “reduced toxicity” means that the toxic or deleteriouseffects upon administration of the virus to a host are attenuated orlessened compared to a host not treated with the virus or compared to ahost that is administered with another reference or control virus. Forpurposes herein, exemplary of a reference or control virus is the LIVPvirus designated GLV-1h68. Whether toxicity is reduced or lessened canbe determined by assessing the effect of a virus and, if necessary, acontrol or reference virus, on a parameter indicative of toxicity. It isunderstood that when comparing the activity of two or more differentviruses, the amount of virus (e.g. pfu) used in an in vitro assay oradministered in vivo is the same or similar and the conditions (e.g. invivo dosage regime) of the in vitro assay or in vivo assessment are thesame or similar. For example, when comparing effects upon in vivoadministration of a virus and a control or reference virus the subjectsare the same species, size, gender and the virus is administered in thesame or similar amount under the same or similar dosage regime. Inparticular, a virus with reduced toxicity can mean that uponadministration of the virus to a host, such as for the treatment of adisease, the virus does not accumulate in non-tumorous organs andtissues in the host to an extent that results in damage or harm to thehost, or that impacts survival of the host to a greater extent than thedisease being treated does or to a greater extent than a control orreference virus does. For example, a virus with reduced toxicityincludes a virus that does not result in death of the subject over thecourse of treatment.

As used herein, accumulation of a virus in a particular tissue refers tothe distribution of the virus in particular tissues of a host organismafter a time period following administration of the virus to the host,long enough for the virus to infect the host's organs or tissues. As oneskilled in the art will recognize, the time period for infection of avirus will vary depending on the virus, the organ(s) or tissue(s), theimmunocompetence of the host and dosage of the virus. Generally,accumulation can be determined at time points from about less than 1day, about 1 day to about 2, 3, 4, 5, 6 or 7 days, about 1 week to about2, 3 or 4 weeks, about 1 month to about 2, 3, 4, 5, 6 months or longerafter infection with the virus. For purposes herein, the virusespreferentially accumulate in immunoprivileged tissue, such as inflamedtissue or tumor tissue, but are cleared from other tissues and organs,such as non-tumor tissues, in the host to the extent that toxicity ofthe virus is mild or tolerable and at most, not fatal.

As used herein, “preferential accumulation” refers to accumulation of avirus at a first location at a higher level than accumulation at asecond location (i.e., the concentration of viral particles, or titer,at the first location is higher than the concentration of viralparticles at the second location). Thus, a virus that preferentiallyaccumulates in immunoprivileged tissue (tissue that is sheltered fromthe immune system), such as inflamed tissue, and tumor tissue, relativeto normal tissues or organs, refers to a virus that accumulates inimmunoprivileged tissue, such as tumor, at a higher level (i.e.,concentration or viral titer) than the virus accumulates in normaltissues or organs.

As used herein, “anti-tumor activity” or “anti-tumorigenic” refers tovirus strains that prevent or inhibit the formation or growth of tumorsin vitro or in vivo in a subject. Anti-tumor activity can be determinedby assessing a parameter or parameters indicative of anti-tumoractivity.

As used herein, a “parameter indicative of anti-tumor activity oranti-tumorigenic activity” refers to a property mediated by a virus thatis associated with anti-tumor activity. Parameters indicative ofanti-tumor activity can be assessed in vitro or in vivo uponadministration to a subject. Exemplary parameters indicative ofanti-tumor activity include, but are not limited to, infectivity oftumor cells, accumulation of virus in tumor tissues, viral nucleic acidreplication in tumor cells, virus production in tumor cells, viral geneexpression in tumor cells, cytotoxicity of tumor cells, tumor cellselectivity, tumor cell type selectivity, decreased tumor size,increased tumor volume, decreased tumor weight, and initiation ofspecific and nonspecific anti-tumor immune responses. Assays that assessany of the above parameters or other anti-tumorigenic properties areknown to one of skill in the art. Hence, a virus that exhibits any oneor more of the above activities or properties exhibits anti-tumoractivity.

As used herein, “greater” or “improved” activity with reference toanti-tumor activity or anti-tumorigenicity means that a virus strain iscapable of preventing or inhibiting the formation or growth of tumors invitro or in vivo in a subject to a greater extent than a reference orcontrol virus or to a greater extent than absence of treatment with thevirus. For purposes herein, exemplary of a reference or control virus isthe LIVP virus designated GLV-1h68. Whether anti-tumor activity is“greater” or “improved” can be determined by assessing the effect of avirus and, if necessary, a control or reference virus, on a parameterindicative of anti-tumor activity. It is understood that when comparingthe activity of two or more different viruses, the amount of virus (e.g.pfu) used in an in vitro assay or administered in vivo is the same orsimilar, and the conditions (e.g. in vivo dosage regime) of the in vitroassay or in vivo assessment are the same or similar.

As used herein, “genetic therapy” or “gene therapy” refers toadministration of a nucleic acid that encodes for a biologicaltherapeutic. Genetic therapy involves the transfer of heterologousnucleic acid, such as DNA, into certain cells, target cells, of amammal, particularly a human, with a disorder or conditions for whichsuch therapy is sought. The nucleic acid, such as DNA, is introducedinto the selected target cells, such as directly or in a vector or otherdelivery vehicle, in a manner such that the heterologous nucleic acid,such as DNA, is expressed and a therapeutic product encoded thereby isproduced. Alternatively, the heterologous nucleic acid, such as DNA, canin some manner mediate expression of DNA that encodes the therapeuticproduct, or it can encode a product, such as a peptide or RNA that insome manner mediates, directly or indirectly, expression of atherapeutic product. Genetic therapy also can be used to deliver nucleicacid encoding a gene product that replaces a defective gene orsupplements a gene product produced by the mammalian or the cell inwhich it is introduced. The introduced nucleic acid can encode atherapeutic compound, such as a growth factor inhibitor thereof, or atumor necrosis factor or inhibitor thereof, such as a receptor therefor,that is not normally produced in the mammalian host or that is notproduced in therapeutically effective amounts or at a therapeuticallyuseful time. The heterologous nucleic acid, such as DNA, encoding thetherapeutic product can be modified prior to introduction into the cellsof the afflicted host in order to enhance or otherwise alter the productor expression thereof. Genetic therapy also can involve delivery of aninhibitor or repressor or other modulator of gene expression. In someembodiments, the nucleic acid encodes a reporter protein for detectionin the methods herein. In some embodiments, the nucleic acid iscontained in a vector. The vector can be a viral vector, mammalianvector, bacterial vector, insect vector, plant vector and artificialchromosome. In some embodiments, the vector is administered in aliposome, PEGylated liposome, nanoparticle, lipid-based nanoparticle orlymphocyte. In some embodiments, the vector is delivered or administereddirectly to a subject.

As used herein, immunotherapy refers to treatment of a disease ordisorder by inducing, enhancing or suppressing an immune responsethrough the use of immunomodulators, such as interleukins, cytokines,chemokines, cytosine phosphate-guanosine, oligodeoxynucleotides andglucans, and cells, such as T cells, lymphocytes, macrophages, dendriticcells, natural killer cells and cytotoxic T lymphocytes. Use of suchimmunomodulators or cells supplements, enhances, replaces or otherwisemodify's the subject's own inadequate or inappropriate immune response.Cancer immunotherapy refers to stimulation of the immune system toreject and destroy tumors, for example, with cytokines Activeimmunotherapy involves injection of cells or proteins, for example,cancer or tumor cells, to generate either new or enhance systemic immuneresponses to the administered cell or protein. Passive immunotherapyinvolves the administration of an antibody.

As used herein, adoptive immunotherapy refers to treatment of tumors,cancers or proliferative disorders using cells having anti-tumoractivity. Cells for adoptive immunotherapy include but are not limitedto activated or expanded T cells, lymphocytes, macrophages, dendriticcells, natural killer cells and cytotoxic T lymphocytes. Immune cellsare administered to a host with the aim that the cells mediate eitherdirectly or indirectly specific immunity to tumor cells and/or antigeniccomponents or regression of the tumor or treatment of infectiousdiseases.

As used herein, the terms overproduce or overexpress when used inreference to a substance, molecule, compound or composition made in acell refers to production or expression at a level that is greater thana baseline, normal or usual level of production or expression of thesubstance, molecule, compound or composition by the cell. A baseline,normal or usual level of production or expression includes noproduction/expression or limited, restricted or regulatedproduction/expression. Such overproduction or overexpression istypically achieved by modification of cell.

As used herein, an agent or compound that modulates the activity of aprotein or expression of a gene or nucleic acid either decreases orincreases or otherwise alters the activity of the protein or, in somemanner, up- or down-regulates or otherwise alters expression of thenucleic acid in a cell.

As used herein, a heterologous nucleic acid (also referred to asexogenous nucleic acid or foreign nucleic acid) refers to a nucleic acidthat is not normally produced in vivo by an organism or virus from whichit is expressed or that is produced by an organism or a virus but is ata different locus, or that mediates or encodes mediators that alterexpression of endogenous nucleic acid, such as DNA, by affectingtranscription, translation, or other regulatable biochemical processes.Hence, heterologous nucleic acid is often not normally endogenous to avirus into which it is introduced. Heterologous nucleic acid can referto a nucleic acid molecule from another virus in the same organism oranother organism, including the same species or another species.Heterologous nucleic acid, however, can be endogenous, but is nucleicacid that is expressed from a different locus or altered in itsexpression or sequence (e.g., a plasmid). Thus, heterologous nucleicacid includes a nucleic acid molecule not present in the exactorientation or position as the counterpart nucleic acid molecule, suchas DNA, is found in a genome. Generally, although not necessarily, suchnucleic acid encodes RNA and proteins that are not normally produced bythe virus or in the same way in the virus in which it is expressed. Anynucleic acid, such as DNA, that one of skill in the art recognizes orconsiders as heterologous, exogenous or foreign to the virus in whichthe nucleic acid is expressed is herein encompassed by heterologousnucleic acid. Examples of heterologous nucleic acid include, but are notlimited to, nucleic acid that encodes exogenous peptides/proteins,including diagnostic and/or therapeutic agents. Proteins that areencoded by heterologous nucleic acid can be expressed within the virus,secreted, or expressed on the surface of the virus in which theheterologous nucleic acid has been introduced.

As used herein, an endogenous nucleic acid or protein refers to anucleic acid or protein that is native to the organism or virus fromwhich it is expressed.

As used herein, a viral clonal strain or virus strain preparation thatcontains heterologous nucleic acid refers to such strains that containnucleic acid not present in the parental clonal strain. For example, thevirus whose sequence is set forth in SEQ ID NO: 91 is a clonal strain,but the virus of SEQ ID NO: 90, designated GLV-1h68, containsheterologous nucleic acid, such as the insert designated RUC-GFP.

As used herein, a heterologous protein or heterologous polypeptide (alsoreferred to as exogenous protein, exogenous polypeptide, foreign proteinor foreign polypeptide) refers to a protein that is not normallyproduced by host, such as the virus.

As used herein, “operably” or “operatively linked” when referring to DNAsegments means that the segments are arranged so that they function inconcert for their intended purposes, e.g., transcription initiatesdownstream of the promoter and upstream of any transcribed sequences.The promoter is usually the domain to which the transcriptionalmachinery binds to initiate transcription and proceeds through thecoding segment to the terminator. “Operative linkage” of heterologousnucleic acids to regulatory and effector sequences of nucleotides, suchas promoters, enhancers, transcriptional and translational stop sites,and other signal sequences refers to the relationship between suchnucleic acid, such as DNA, and such sequences of nucleotides. Forexample, operative linkage of heterologous DNA to a promoter refers tothe physical relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. Thus, operatively linked or operationally associated refers to thefunctional relationship of a nucleic acid, such as DNA, with regulatoryand effector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signalsequences. For example, operative linkage of DNA to a promoter refers tothe physical and functional relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA. In order to optimize expression and/ortranscription, it can be necessary to remove, add or alter 5′untranslated portions of the clones to eliminate extra, potentiallyinappropriate, alternative translation initiation (i.e., start) codonsor other sequences that can interfere with or reduce expression, eitherat the level of transcription or translation. In addition, consensusribosome binding sites can be inserted immediately 5′ of the start codonand can enhance expression (see, e.g., Kozak J. Biol. Chem. 266:19867-19870 (1991) and Shine and Delgarno, Nature 254(5495):34-38(1975)). The desirability of (or need for) such modification can beempirically determined.

As used herein, a heterologous promoter refers to a promoter that is notnormally found in the wild-type organism or virus or that is at adifferent locus as compared to a wild-type organism or virus. Aheterologous promoter is often not endogenous to a virus into which itis introduced, but has been obtained from another virus or preparedsynthetically. A heterologous promoter can refer to a promoter fromanother virus in the same organism or another organism, including thesame species or another species. A heterologous promoter, however, canbe endogenous, but is a promoter that is altered in its sequence oroccurs at a different locus (e.g., at a different location in the genomeor on a plasmid). Thus, a heterologous promoter includes a promoter notpresent in the exact orientation or position as the counterpart promoteris found in a genome.

A synthetic promoter is a heterologous promoter that has a nucleotidesequence that is not found in nature. A synthetic promoter can be anucleic acid molecule that has a synthetic sequence or a sequencederived from a native promoter or portion thereof. A synthetic promoteralso can be a hybrid promoter composed of different elements derivedfrom different native promoters.

As used herein, vector (or plasmid) refers to a nucleic acid constructthat contains discrete elements that are used to introduce heterologousnucleic acid into cells for either expression of the nucleic acid orreplication thereof. The vectors typically remain episomal, but can bedesigned to effect stable integration of a gene or portion thereof intoa chromosome of the genome. Selection and use of such vectors are wellknown to those of skill in the art. Expression vectors include vectorscapable of expressing DNA that is operatively linked with regulatorysequences, such as promoter regions, that are capable of effectingexpression of the DNA fragments. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome. A vector can be a viral vector,mammalian vector, bacterial vector, insect vector and plant vector. Alsocontemplated are vectors that are artificial chromosomes, such as yeastartificial chromosomes and mammalian artificial chromosomes. In someembodiments, the vector is administered in a liposome, PEGylatedliposome, nanoparticle, lipid-based nanoparticle or lymphocyte. In someembodiments, the vector is delivered or administered directly to asubject. Selection and use of such vehicles are well known to those ofskill in the art.

As used herein, the term “viral vector” is used according to itsart-recognized meaning. It refers to a nucleic acid vector that includesat least one element of viral origin and can be packaged into a viralvector particle. The viral vector particles can be used for the purposeof transferring DNA, RNA or other nucleic acids into cells either invitro or in vivo. Viral vectors include, but are not limited to,poxvirus vectors (e.g., vaccinia vectors), retroviral vectors,lentivirus vectors, herpes virus vectors (e.g., HSV), baculovirusvectors, cytomegalovirus (CMV) vectors, papillomavirus vectors, simianvirus (SV40) vectors, semliki forest virus vectors, phage vectors,adenoviral vectors and adeno-associated viral (AAV) vectors.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. Nucleic acids can encode geneproducts, such as, for example, polypeptides, regulatory RNAs,microRNAs, siRNAs and functional RNAs.

As used herein, a sequence complementary to at least a portion of anRNA, with reference to antisense oligonucleotides, means a sequence ofnucleotides having sufficient complementarity to be able to hybridizewith the RNA, generally under moderate or high stringency conditions,forming a stable duplex; in the case of double-stranded antisensenucleic acids, a single strand of the duplex DNA (i.e., dsRNA) can thusbe assayed, or triplex formation can be assayed. The ability tohybridize depends on the degree of complementarity and the length of theantisense nucleic acid. Generally, the longer the hybridizing nucleicacid, the more base mismatches with an encoding RNA it can contain andstill form a stable duplex (or triplex, as the case can be). One skilledin the art can ascertain a tolerable degree of mismatch by use ofstandard procedures to determine the melting point of the hybridizedcomplex.

As used herein, a substrate refers to an atom, molecule, compound orcomposition that is a reactant for a reporter protein. For example, asubstrate for a reporter enzyme is a compound that is consumed duringthe catalytic or enzymatic reaction or a molecule that is acted upon bythe enzyme. Typically substrates or detectable or generate a detectableproduct upon interaction with the reporter protein. For example,substrates include fluorescent, luminescent, spectrophotometric,fluorogenic, chromogenic and radioactive substrates. Such substrates canbe detected, for example, by visual inspection, with aspectrophotometer, fluorometer, luminometer, scintillation counter orRaman spectrometer, by reflectance measurement, by flow cytometry and byX-rays. Substrates also include contrast agents or alternatively asubstrate can generate a contrast agent for use in PET imaging.

As used herein, beta-glucuronidase, β-glucuronidase or gusA refers toenzymes that catalyze the hydrolysis of β-D-glucuronides.Beta-glucuronidases include any of non-human origin including, but notlimited to, beta-glucuronidases from mouse (SEQ ID NO:115, DNA set forthin SEQ ID NO:2), rat (SEQ ID NO:114, DNA set forth in SEQ ID NO:6), dog(SEQ ID NO:116, DNA set forth in SEQ ID NO:7), cat (SEQ ID NO:117, DNAset forth in SEQ ID NO:8), pig (SEQ ID NO:120, DNA set forth in SEQ IDNO:11), green monkey (SEQ ID NO:118, DNA set forth in SEQ ID NO:9) andSumatran orangutan (SEQ ID NO:119, DNA set forth in SEQ ID NO:10).Beta-glucuronidases also include those of human origin. Exemplary of ahuman beta-glucuronidase is the human beta-glucuronidase set forth inSEQ ID NOS:5 and 121. Beta-glucuronidases also include bacterialbeta-glucuronidases, such as beta-glucuronidases from E. coli K12 (SEQID NO:4, DNA set forth in SEQ ID NO:3), Shigella flexneri strain K-18(SEQ ID NO:128, DNA set forth in SEQ ID NO:127), Salmonella enterica(SEQ ID NO:136, DNA set forth in SEQ ID NO:135), Lactobacillus brevisstrain RO1 (SEQ ID NO:130, DNA set forth in SEQ ID NO:129),Streptococcus agalactiae (SEQ ID NO:132, DNA set forth in SEQ IDNO:131), Clostridium perfringens (SEQ ID NO:134, DNA set forth in SEQ IDNO:133), Roseburia intestinalis (SEQ ID NO:138, DNA set forth in SEQ IDNO:137), Anaerococcus tetradius (SEQ ID NO:140, DNA set forth in SEQ IDNO:139), Victivallis vadensis (SEQ ID NO:142, DNA set forth in SEQ IDNO:141), Congregibacter litoralis (SEQ ID NO:144, DNA set forth in SEQID NO:143) and Aspergillus terreus (SEQ ID NO:146, DNA set forth in SEQID NO:145). Exemplary of a bacterial beta-glucuronidase is the E. colibeta-glucuronidase set forth in SEQ ID NO:4. Reference tobeta-glucuronidases includes allelic and species variants, truncatedforms that have activity, splice variants and other variants, includingbeta-glucuronidases that have at least 60%, 70%, 80%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thebeta-glucuronidases set forth in SEQ ID NOS: 1-11, 114-121, 127-146.

As used herein, beta-galactosidase, β-galactosidase or lacZ refers toenzymes that catalyze the hydrolysis of β-galactosides intomonosaccharides. Beta-galactosidases include any of non-human originincluding, but not limited to, mouse (SEQ ID NO:29, DNA set forth in SEQID NO:28) and dog (SEQ ID NO:33, DNA set forth in SEQ ID NO:32).Beta-galactosidases also include those of human origin. Exemplary of ahuman beta-galactosidase is the human beta-galactosidase set forth inSEQ ID NOS:13 and 122. Beta-galactosidases also include bacterialbeta-glucuronidases, such as beta-galactosidases from E. coli K12 (SEQID NO:14, DNA set forth in SEQ ID NO:15), Lactobacillus acidophilus (SEQID NO:16, DNA set forth in SEQ ID NO:17), Sulfolobus solfataricus (SEQID NO:18, DNA set forth in SEQ ID NO:19), Lactococcus lactis (SEQ IDNO:20, DNA set forth in SEQ ID NO:21), Geobacillus kaustiophilus (SEQ IDNO:22, DNA set forth in SEQ ID NO:23), Thermus thermophilus (SEQ IDNO:24, DNA set forth in SEQ ID NO:25), Bacillus subtilis (SEQ ID NO:26,DNA set forth in SEQ ID NO:27) and Clostridium perfringens (SEQ IDNO:30, DNA set forth in SEQ ID NO:31). Exemplary of a β-galactosidase ishuman β-galactosidase (set forth in SEQ ID NO:13). Exemplary of abacterial beta-galactosidase is the E. coli beta-galactosidase set forthin SEQ ID NO:14. Reference to beta-glucuronidases includes allelic andspecies variants, truncated forms that have activity, splice variantsand other variants, including beta-glucuronidases that have at least60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to the beta-galactosidases set forth in SEQ ID NOS:12-33 and 122.

As used herein, chloramphenicol acetyltransferases or CAT refers tobacterial enzymes (EC 2.3.1.28) that detoxify the antibioticchloramphenicol. Chloramphenicol acetyltransferases include, but are notlimited to, those from E. coli strain DJ33-16 (SEQ ID NO:53, DNA setforth in SEQ ID NO:52), Pseudomonas aeruginosa (SEQ ID NO:55, DNA setforth in SEQ ID NO:54), Staphylococcus aureus (SEQ ID NO:57, DNA setforth in SEQ ID NO:56), Agrobacterium tumefaciens (SEQ ID NO:59, DNA setforth in SEQ ID NO:58), Clostridium perfingens (SEQ ID NO:61, DNA setforth in SEQ ID NO:60), Klebsiella pneumoniae (SEQ ID NO:63, DNA setforth in SEQ ID NO:62), Haemophilus influenzae (SEQ ID NO:65, DNA setforth in SEQ ID NO:64), Streptococcus agalactiae (SEQ ID NO:67, DNA setforth in SEQ ID NO:66), Bacillus pumilus (SEQ ID NO:69, DNA set forth inSEQ ID NO:68), Proteus mirabilis (SEQ ID NO:71, DNA set forth in SEQ IDNO:70), Salmonella enterica (SEQ ID NO:73, DNA set forth in SEQ IDNO:72), Staphylococcus intermedius (SEQ ID NO:75, DNA set forth in SEQID NO:74), Listonella anguillarum (SEQ ID NO:77, DNA set forth in SEQ IDNO:76), Campylobacter coli (SEQ ID NO:79, DNA set forth in SEQ ID NO:78)and Acinetobacter baumannii (SEQ ID NO:81, DNA set forth in SEQ IDNO:80). Reference to chloramphenicol acetyltransferases includes allelicand species variants, truncated forms that have activity, splicevariants and other variants, including chloramphenicolacetyltransferases that have at least 60%, 70%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thechloramphenicol acetyltransferases set forth in SEQ ID NOS:52-81.

As used herein, alkaline phosphatases refers to hydrolase enzymesresponsible for removing phosphate groups, e.g., dephosphorylating, frommany types of molecules, including nucleotides, proteins, and alkaloids.Alkaline phosphatases are most effective in an alkaline environment.Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate estersin alkaline buffer and produces an organic radical and inorganicphosphate. Alkaline phosphatases include, but are not limited to, shrimpalkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalusborealis) (SEQ ID NO:109, DNA set forth in SEQ ID NO:108), IntestinalAlkaline Phosphatase (AIP; SEQ ID NO:111, DNA set forth in SEQ IDNO:110) and Placental alkaline phosphatase (PALP; SEQ ID NOS:113, DNAset forth in SEQ ID NO:99 and 112) and secreted alkaline phosphatase(SEAP; SEQ ID NO:126), a C terminally truncated version of PALP.Reference to alkaline phosphatases includes allelic and speciesvariants, truncated forms that have activity, splice variants and othervariants, including alkaline phosphatases that have at least 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to the alkaline phosphatases set forth in SEQ ID NOS:108-113and 126.

As used herein, luminescence refers to the detectable electromagnetic(EM) radiation, generally, ultraviolet (UV), infrared (IR) or visible EMradiation that is produced when the excited product of an exergonicchemical process reverts to its ground state with the emission of light.Chemiluminescence is luminescence that results from a chemical reaction.Bioluminescence is chemiluminescence that results from a chemicalreaction using biological molecules (or synthetic versions or analogsthereof) as substrates and/or enzymes. Fluorescence is luminescence inwhich light of a visible color is emitted from a substance understimulation or excitation by light or other forms radiation such asultraviolet (UV), infrared (IR) or visible EM radiation.

As used herein, chemiluminescence refers to a chemical reaction in whichenergy is specifically channeled to a molecule causing it to becomeelectronically excited and subsequently to release a photon, therebyemitting visible light. Temperature does not contribute to thischanneled energy. Thus, chemiluminescence involves the direct conversionof chemical energy to light energy.

As used herein, bioluminescence, which is a type of chemiluminescence,refers to the emission of light by biological molecules, particularlyproteins. The essential condition for bioluminescence is molecularoxygen, either bound or free in the presence of an oxygenase, aluciferase, which acts on a substrate, a luciferin. Bioluminescence isgenerated by an enzyme or other protein (luciferase) that is anoxygenase that acts on a substrate luciferin (a bioluminescencesubstrate) in the presence of molecular oxygen and transforms thesubstrate to an excited state, which, upon return to a lower energylevel releases the energy in the form of light.

As used herein, the substrates and enzymes for producing bioluminescenceare generically referred to as luciferin and luciferase, respectively.When reference is made to a particular species thereof, for clarity,each generic term is used with the name of the organism from which itderives such as, for example, click beetle luciferase or fireflyluciferase.

As used herein, luciferase refers to oxygenases that catalyze a lightemitting reaction. For instance, bacterial luciferases catalyze theoxidation of flavin mononucleotide (FMN) and aliphatic aldehydes, whichreaction produces light. Another class of luciferases, found amongmarine arthropods, catalyzes the oxidation of Cypridina (Vargula)luciferin and another class of luciferases catalyzes the oxidation ofColeoptera luciferin. Thus, luciferase refers to an enzyme orphotoprotein that catalyzes a bioluminescent reaction (a reaction thatproduces bioluminescence). The luciferases, such as firefly and Gaussiaand Renilla luciferases, are enzymes which act catalytically and areunchanged during the bioluminescence generating reaction. The luciferasephotoproteins, such as the aequorin photoprotein to which luciferin isnon-covalently bound, are changed, such as by release of the luciferin,during bioluminescence generating reaction. The luciferase is a protein,or a mixture of proteins (e.g., bacterial luciferase), that occursnaturally in an organism or a variant or mutant thereof, such as avariant produced by mutagenesis that has one or more properties, such asthermal stability, that differ from the naturally-occurring protein.Luciferases and modified mutant or variant forms thereof are well known.For purposes herein, reference to luciferase refers to either thephotoproteins or luciferases.

Reference, for example, to Renilla luciferase refers to an enzymeisolated from member of the genus Renilla or an equivalent moleculeobtained from any other source, such as from another related copepod, orthat has been prepared synthetically. It is intended to encompassRenilla luciferases with conservative amino acid substitutions that donot substantially alter activity. Conservative substitutions of aminoacids are known to those of skill in this art and can be made generallywithout altering the biological activity of the resulting molecule.Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224).

As used herein, bioluminescence substrate refers to the compound that isoxidized in the presence of a luciferase and any necessary activatorsand generates light. These substrates are referred to as luciferinsherein, are substrates that undergo oxidation in a bioluminescencereaction. These bioluminescence substrates include any luciferin oranalog thereof or any synthetic compound with which a luciferaseinteracts to generate light. Typical substrates include those that areoxidized in the presence of a luciferase or protein in alight-generating reaction. Bioluminescence substrates, thus, includethose compounds that those of skill in the art recognize as luciferins.Luciferins, for example, include firefly luciferin, Cypridina (alsoknown as Vargula) luciferin (coelenterazine), bacterial luciferin, aswell as synthetic analogs of these substrates or other compounds thatare oxidized in the presence of a luciferase in a reaction that producesbioluminescence.

As used herein, capable of conversion into a bioluminescence substraterefers to being susceptible to chemical reaction, such as oxidation orreduction, which yields a bioluminescence substrate. For example, theluminescence producing reaction of bioluminescent bacteria involves thereduction of a flavin mononucleotide group (FMN) to reduced flavinmononucleotide (FMNH₂) by a flavin reductase enzyme. The reduced flavinmononucleotide (substrate) then reacts with oxygen (an activator) andbacterial luciferase to form an intermediate peroxy flavin thatundergoes further reaction, in the presence of a long-chain aldehyde, togenerate light. With respect to this reaction, the reduced flavin andthe long chain aldehyde are bioluminescence substrates.

As used herein, a bioluminescence generating system refers to the set ofreagents required to conduct a bioluminescent reaction. Thus, thespecific luciferase, luciferin and other substrates, solvents and otherreagents that can be required to complete a bioluminescent reaction forma bioluminescence system. Thus a bioluminescence generating systemrefers to any set of reagents that, under appropriate reactionconditions, yield bioluminescence. Appropriate reaction conditions referto the conditions necessary for a bioluminescence reaction to occur,such as pH, salt concentrations and temperature. In general,bioluminescence systems include a bioluminescence substrate, luciferin,a luciferase, which includes enzymes luciferases and photoproteins andone or more activators. A specific bioluminescence system can beidentified by reference to the specific organism from which theluciferase derives; for example, the Renilla bioluminescence systemincludes a Renilla luciferase, such as a luciferase isolated fromRenilla or produced using recombinant methods or modifications of theseluciferases. This system also includes the particular activatorsnecessary to complete the bioluminescence reaction, such as oxygen and asubstrate with which the luciferase reacts in the presence of the oxygento produce light.

As used herein, a fluorescent protein (FP) refers to a protein thatpossesses the ability to fluoresce (i.e., to absorb energy at onewavelength and emit it at another wavelength). For example, a greenfluorescent protein (GFP) refers to a polypeptide that has a peak in theemission spectrum at 510 nm or about 510 nm. A variety of FPs that emitat various wavelengths are known in the art. Exemplary FPs include, butare not limited to, a green fluorescent protein (GFP), yellowfluorescent protein (YFP), orange fluorescent protein (OFP), cyanfluorescent protein (CFP), blue fluorescent protein (BFP), redfluorescent protein (RFP), far-red fluorescent protein, or near-infraredfluorescent protein. Extending the spectrum of available colors offluorescent proteins to blue, cyan, orange, yellow and red variantsprovides a method for multicolor tracking of fusion proteins.

As used herein, Aequorea GFP refers to GFPs from the genus Aequorea andto mutants or variants thereof. Such variants and GFPs from otherspecies, such as Anthozoa reef coral, Anemonia sea anemone, Renilla seapansy, Galaxea coral, Acropora brown coral, Trachyphyllia andPectiniidae stony coral and other species are well known and areavailable and known to those of skill in the art. Exemplary GFP variantsinclude, but are not limited to BFP, CFP, YFP and OFP. Examples offluorescent proteins and their variants include GFP proteins, such asEmerald (Invitrogen, Carlsbad, Calif.), EGFP (Clontech, Palo Alto,Calif.), Azami-Green (MBL International, Woburn, Mass.), Kaede (MBLInternational, Woburn, Mass.), ZsGreenl (Clontech, Palo Alto, Calif.)and CopGFP (Evrogen/Axxora, LLC, San Diego, Calif.); CFP proteins, suchas Cerulean (Rizzo, Nat Biotechnol. 22(4):445-9 (2004)), mCFP (Wang etal., PNAS U.S.A. 101(48):16745-9 (2004)), AmCyan1 (Clontech, Palo Alto,Calif.), MiCy (MBL International, Woburn, Mass.), and CyPet (Nguyen andDaugherty, Nat Biotechnol. 23(3):355-60 (2005)); BFP proteins such asEBFP (Clontech, Palo Alto, Calif.); YFP proteins such as EYFP (Clontech,Palo Alto, Calif.), YPet (Nguyen and Daugherty, Nat Biotechnol.23(3):355-60 (2005)), Venus (Nagai et al., Nat. Biotechnol. 20(1):87-90(2002)), ZsYellow (Clontech, Palo Alto, Calif.), and mCitrine (Wang etal., PNAS USA. 101(48):16745-9 (2004)); OFP proteins such as cOFP(Stratagene, La Jolla, Calif.), mKO (MBL International, Woburn, Mass.),and mOrange; and others (see, e.g., Shaner N C, Steinbach P A, and TsienR Y., Nat Methods. 2(12):905-9 (2005)).

As used herein, red fluorescent protein, or RFP, refers to the DiscosomaRFP (DsRed) that has been isolated from the corallimorph Discosoma (Matzet al., Nature Biotechnology 17: 969-973 (1999)), and red or far-redfluorescent proteins from any other species, such as Heteractis reefcoral and Actinia or Entacmaea sea anemone, as well as variants thereof.RFPs include, for example, Discosoma variants, such as monomeric redfluorescent protein 1 (mRFP1), mCherry, tdTomato, mStrawberry,mTangerine (Wang et al., PNAS USA. 101(48):16745-9 (2004)), DsRed2(Clontech, Palo Alto, Calif.), and DsRed-T1 (Bevis and Glick, Nat.Biotechnol., 20: 83-87 (2002)), Anthomedusa J-Red (Evrogen) and AnemoniaAsRed2 (Clontech, Palo Alto, Calif.). Far-red fluorescent proteinsinclude, for example, Actinia AQ143 (Shkrob et al., Biochem J. 392(Pt3):649-54 (2005)), Entacmaea eqFP611 (Wiedenmann et al. Proc. Natl.Acad. Sci. USA. 99(18):11646-51 (2002)), Discosoma variants such asmPlum and mRasberry (Wang et al., PNAS USA. 101(48):16745-9 (2004)), andHeteractis HcRed1 and t-HcRed (Clontech, Palo Alto, Calif.).Near-infrared fluorescent proteins include, for example, mKate,TurboFP635 or Katushka, mNeptune and IFP1.4 (Shcherbo et al., (2007) NatMethods 4:741-746).

As used herein, antibody refers to monoclonal antibodies, polyclonalantibodies, and chimeric antibodies. Antibodies and immunoglobulins areglycoproteins having the same structural characteristics. Nativeantibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each heavy chain has atone end a variable domain (V_(H)) followed by a number of constantdomains. Each light chain has a variable domain at one end (V_(L)) and aconstant domain at its other end. The antibody may be from recombinantsources and/or produced in transgenic animals. Antibodies include Fab,Fab′, F(ab′)₂, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, andmultimers thereof and multispecific antibody fragments.

As used herein, an antigen is a substance that evokes the production ofone or more antibodies. Antigens are characterized by their ability tobe “bound” in the antigen-binding site of an antibody. Antigens includeproteins and polysaccharides, such as those from bacteria, viruses andmicroorganism.

As used herein, nanoparticle refers to a microscopic particle whose sizeis measured in nanometers. Often such particles in nanoscale are used inbiomedical applications acting as drug carriers or imaging agents.Nanoparticles can be conjugated to other agents, including, but notlimited to detectable/diagnostic agents or therapeutic agents.

As used herein, an in vivo method refers to a method performed withinthe living body of a subject.

As used herein, “nucleic acids” include DNA, RNA and analogs thereof,including peptide nucleic acids (PNA) and mixtures thereof. Nucleicacids can be single or double-stranded. When referring to probes orprimers, which are optionally labeled, such as with a detectable label,such as a fluorescent or radiolabel, single-stranded molecules arecontemplated. Such molecules are typically of a length such that theirtarget is statistically unique or of low copy number (typically lessthan 5, generally less than 3) for probing or priming a library.Generally a probe or primer contains at least 14, 16 or 30 contiguousnucleotides of sequence complementary to or identical to a gene ofinterest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleicacids long.

As used herein, a peptide refers to a polypeptide that is greater thanor equal to 2 amino acids in length, and less than or equal to 40 aminoacids in length.

As used herein, the amino acids which occur in the various sequences ofamino acids provided herein are identified according to their known,three-letter or one-letter abbreviations (Table 1). The nucleotideswhich occur in the various nucleic acid fragments are designated withthe standard single-letter designations used routinely in the art.

As used herein, an “amino acid” is an organic compound containing anamino group and a carboxylic acid group. A polypeptide contains two ormore amino acids. For purposes herein, amino acids include the twentynaturally-occurring amino acids, non-natural amino acids and amino acidanalogs (i.e., amino acids wherein the α-carbon has a side chain).

As used herein, “amino acid residue” refers to an amino acid formed uponchemical digestion (hydrolysis) of a polypeptide at its peptidelinkages. The amino acid residues described herein are presumed to be inthe “L” isomeric form. Residues in the “D” isomeric form, which are sodesignated, can be substituted for any L-amino acid residue as long asthe desired functional property is retained by the polypeptide. NH2refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxy group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), andadopted 37 C.F.R. §§1.821-1.822, abbreviations for amino acid residuesare shown in Table 1:

TABLE 1 Table of Correspondence SYMBOL 1-Letter 3-Letter AMINO ACID YTyr Tyrosine G Gly Glycine F Phe Phenylanine M Met Methionine A AlaAlanine S Ser Serine I Ile Isoleucine L Leu Leucine T Thr Threonine VVal Valine P Pro Proline K Lys Lysine H His Histidine Q Gln Glutamine EGlu Glutamic acid Z Glx Glu and/or Gln W Trp Tryptophan R Arg Arginine DAsp Aspartic acid N Asn Asparagine B Asx Asn and/or Asp C Cys Cysteine XXaa Unknown or other

All amino acid residue sequences represented herein by formulae have aleft to right orientation in the conventional direction ofamino-terminus to carboxyl-terminus. In addition, the phrase “amino acidresidue” is defined to include the amino acids listed in the Table ofCorrespondence (Table 1) and modified and unusual amino acids, such asthose referred to in 37 C.F.R. §§1.821-1.822, and incorporated herein byreference. Furthermore, a dash at the beginning or end of an amino acidresidue sequence indicates a peptide bond to a further sequence of oneor more amino acid residues, to an amino-terminal group such as NH₂ orto a carboxyl-terminal group such as COOH.

As used herein, the “naturally occurring α-amino acids” are the residuesof those 20 α-amino acids found in nature which are incorporated intoprotein by the specific recognition of the charged tRNA molecule withits cognate mRNA codon in humans. Non-naturally occurring amino acidsthus include, for example, amino acids or analogs of amino acids otherthan the 20 naturally-occurring amino acids and include, but are notlimited to, the D-isostereomers of amino acids. Exemplary non-naturalamino acids are described herein and are known to those of skill in theart.

As used herein, a DNA construct is a single- or double-stranded, linearor circular DNA molecule that contains segments of DNA combined andjuxtaposed in a manner not found in nature. DNA constructs exist as aresult of human manipulation, and include clones and other copies ofmanipulated molecules.

As used herein, a DNA segment is a portion of a larger DNA moleculehaving specified attributes. For example, a DNA segment encoding aspecified polypeptide is a portion of a longer DNA molecule, such as aplasmid or plasmid fragment, which, when read from the 5′ to 3′direction, encodes the sequence of amino acids of the specifiedpolypeptide.

As used herein, the term polynucleotide means a single- ordouble-stranded polymer of deoxyribonucleotides or ribonucleotide basesread from the 5′ to the 3′ end. Polynucleotides include RNA and DNA, andcan be isolated from natural sources, synthesized in vitro, or preparedfrom a combination of natural and synthetic molecules. The length of apolynucleotide molecule is given herein in terms of nucleotides(abbreviated “nt”) or base pairs (abbreviated “bp”). The termnucleotides is used for single- and double-stranded molecules where thecontext permits. When the term is applied to double-stranded moleculesit is used to denote overall length and will be understood to beequivalent to the term base pairs. It will be recognized by thoseskilled in the art that the two strands of a double-strandedpolynucleotide can differ slightly in length and that the ends thereofcan be staggered; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will, ingeneral, not exceed 20 nucleotides in length.

As used herein, “similarity” between two proteins or nucleic acidsrefers to the relatedness between the sequence of amino acids of theproteins or the nucleotide sequences of the nucleic acids. Similaritycan be based on the degree of identity and/or homology of sequences ofresidues and the residues contained therein. Methods for assessing thedegree of similarity between proteins or nucleic acids are known tothose of skill in the art. For example, in one method of assessingsequence similarity, two amino acid or nucleotide sequences are alignedin a manner that yields a maximal level of identity between thesequences. “Identity” refers to the extent to which the amino acid ornucleotide sequences are invariant. Alignment of amino acid sequences,and to some extent nucleotide sequences, also can take into accountconservative differences and/or frequent substitutions in amino acids(or nucleotides). Conservative differences are those that preserve thephysico-chemical properties of the residues involved. Alignments can beglobal (alignment of the compared sequences over the entire length ofthe sequences and including all residues) or local (the alignment of aportion of the sequences that includes only the most similar region orregions).

“Identity” per se has an art-recognized meaning and can be calculatedusing published techniques. (See, e.g. Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). While there exists a number of methodsto measure identity between two polynucleotide or polypeptides, the term“identity” is well known to skilled artisans (Carrillo, H. & Lipton, D.,SIAM J Applied Math 48:1073 (1988)).

As used herein, homologous (with respect to nucleic acid and/or aminoacid sequences) means about greater than or equal to 25% sequencehomology, typically greater than or equal to 25%, 40%, 50%, 60%, 70%,80%, 85%, 90% or 95% sequence homology; the precise percentage can bespecified if necessary. For purposes herein the terms “homology” and“identity” are often used interchangeably, unless otherwise indicated.In general, for determination of the percentage homology or identity,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carrillo et al. (1988) SIAM J Applied Math 48:1073). By sequencehomology, the number of conserved amino acids is determined by standardalignment algorithms programs, and can be used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules hybridize typically at moderate stringency or at highstringency all along the length of the nucleic acid of interest. Alsocontemplated are nucleic acid molecules that contain degenerate codonsin place of codons in the hybridizing nucleic acid molecule.

Whether any two molecules have nucleotide sequences or amino acidsequences that are at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% “identical” or “homologous” can be determined using knowncomputer algorithms such as the “FASTA” program, using for example, thedefault parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.USA 85:2444 (other programs include the GCG program package (Devereux,J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN,FASTA (Atschul, S. F., et al., J Mol Biol 215:403 (1990)); Guide to HugeComputers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, andCarrillo et al. (1988) SIAM J Applied Math 48:1073). For example, theBLAST function of the National Center for Biotechnology Informationdatabase can be used to determine identity. Other commercially orpublicly available programs include, DNAStar “MegAlign” program(Madison, Wis.) and the University of Wisconsin Genetics Computer Group(UWG) “Gap” program (Madison Wis.). Percent homology or identity ofproteins and/or nucleic acid molecules can be determined, for example,by comparing sequence information using a GAP computer program (e.g.,Needleman et al. (1970) J. Mol. Biol. 48:443, as revised by Smith andWaterman ((1981) Adv. Appl. Math. 2:482). Briefly, the GAP programdefines similarity as the number of aligned symbols (i.e., nucleotidesor amino acids), which are similar, divided by the total number ofsymbols in the shorter of the two sequences. Default parameters for theGAP program can include: (1) a unary comparison matrix (containing avalue of 1 for identities and 0 for non-identities) and the weightedcomparison matrix of Gribskov et al. (1986) Nucl. Acids Res. 14:6745, asdescribed by Schwartz and Dayhoff, eds., ATLAS OF PROTEIN SEQUENCE ANDSTRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1979);(2) a penalty of 3.0 for each gap and an additional 0.10 penalty foreach symbol in each gap; and (3) no penalty for end gaps.

Therefore, as used herein, the term “identity” or “homology” representsa comparison between a test and a reference polypeptide orpolynucleotide. As used herein, the term at least “90% identical to”refers to percent identities from 90 to 99.99 relative to the referencenucleic acid or amino acid sequence of the polypeptide. Identity at alevel of 90% or more is indicative of the fact that, assuming forexemplification purposes a test and reference polypeptide length of 100amino acids are compared. No more than 10% (i.e., 10 out of 100) of theamino acids in the test polypeptide differs from that of the referencepolypeptide. Similar comparisons can be made between test and referencepolynucleotides. Such differences can be represented as point mutationsrandomly distributed over the entire length of a polypeptide or they canbe clustered in one or more locations of varying length up to themaximum allowable, e.g. 10/100 amino acid difference (approximately 90%identity). Differences are defined as nucleic acid or amino acidsubstitutions, insertions or deletions. At the level of homologies oridentities above about 85-90%, the result is independent of the programand gap parameters set; such high levels of identity can be assessedreadily, often by manual alignment without relying on software.

As used herein, an aligned sequence refers to the use of homology(similarity and/or identity) to align corresponding positions in asequence of nucleotides or amino acids. Typically, two or more sequencesthat are related by 50% or more identity are aligned. An aligned set ofsequences refers to 2 or more sequences that are aligned atcorresponding positions and can include aligning sequences derived fromRNAs, such as ESTs and other cDNAs, aligned with genomic DNA sequence.

As used herein, “primer” refers to a nucleic acid molecule that can actas a point of initiation of template-directed DNA synthesis underappropriate conditions (e.g., in the presence of four differentnucleoside triphosphates and a polymerization agent, such as DNApolymerase, RNA polymerase or reverse transcriptase) in an appropriatebuffer and at a suitable temperature. It will be appreciated thatcertain nucleic acid molecules can serve as a “probe” and as a “primer.”A primer, however, has a 3′ hydroxyl group for extension. A primer canbe used in a variety of methods, including, for example, polymerasechain reaction (PCR), reverse-transcriptase (RT)-PCR, RNA PCR, LCR,multiplex PCR, panhandle PCR, capture PCR, expression PCR, 3′ and 5′RACE, in situ PCR, ligation-mediated PCR and other amplificationprotocols.

As used herein, “primer pair” refers to a set of primers that includes a5′ (upstream) primer that hybridizes with the 5′ end of a sequence to beamplified (e.g. by PCR) and a 3′ (downstream) primer that hybridizeswith the complement of the 3′ end of the sequence to be amplified.

As used herein, “specifically hybridizes” refers to annealing, bycomplementary base-pairing, of a nucleic acid molecule (e.g. anoligonucleotide) to a target nucleic acid molecule. Those of skill inthe art are familiar with in vitro and in vivo parameters that affectspecific hybridization, such as length and composition of the particularmolecule. Parameters particularly relevant to in vitro hybridizationfurther include annealing and washing temperature, buffer compositionand salt concentration. Exemplary washing conditions for removingnon-specifically bound nucleic acid molecules at high stringency are0.1×SSPE, 0.1% SDS, 65° C., and at medium stringency are 0.2×SSPE, 0.1%SDS, 50° C. Equivalent stringency conditions are known in the art. Theskilled person can readily adjust these parameters to achieve specifichybridization of a nucleic acid molecule to a target nucleic acidmolecule appropriate for a particular application. Complementary, whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, typically with less than 25%,15% or 5% mismatches between opposed nucleotides. If necessary, thepercentage of complementarity will be specified. Typically the twomolecules are selected such that they will hybridize under conditions ofhigh stringency.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, it also is understood that the terms “substantiallyidentical” or “similar” varies with the context as understood by thoseskilled in the relevant art.

As used herein, an allelic variant or allelic variation references anyof two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and can result in phenotypic polymorphism within populations. Genemutations can be silent (no change in the encoded polypeptide) or canencode polypeptides having altered amino acid sequence. The term“allelic variant” also is used herein to denote a protein encoded by anallelic variant of a gene. Typically the reference form of the geneencodes a wildtype form and/or predominant form of a polypeptide from apopulation or single reference member of a species. Typically, allelicvariants, which include variants between and among species typicallyhave at least 80%, 90% or greater amino acid identity with a wildtypeand/or predominant form from the same species; the degree of identitydepends upon the gene and whether comparison is interspecies orintraspecies. Generally, intraspecies allelic variants have at leastabout 80%, 85%, 90% or 95% identity or greater with a wildtype and/orpredominant form, including 96%, 97%, 98%, 99% or greater identity witha wildtype and/or predominant form of a polypeptide. Reference to anallelic variant herein generally refers to variations n proteins amongmembers of the same species.

As used herein, “allele,” which is used interchangeably herein with“allelic variant” refers to alternative forms of a gene or portionsthereof. Alleles occupy the same locus or position on homologouschromosomes. When a subject has two identical alleles of a gene, thesubject is said to be homozygous for that gene or allele. When a subjecthas two different alleles of a gene, the subject is said to beheterozygous for the gene. Alleles of a specific gene can differ fromeach other in a single nucleotide or several nucleotides, and caninclude modifications such as substitutions, deletions and insertions ofnucleotides. An allele of a gene also can be a form of a gene containinga mutation.

As used herein, species variants refer to variants in polypeptides amongdifferent species, including different mammalian species, such as mouseand human. For example for β-glucuronidase, exemplary of speciesvariants provided herein are mouse, rat, cat, dog, pig, green monkey andSumatran orangutan. Generally, species variants have 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity.Corresponding residues between and among species variants can bedetermined by comparing and aligning sequences to maximize the number ofmatching nucleotides or residues, for example, such that identitybetween the sequences is equal to or greater than 95%, equal to orgreater than 96%, equal to or greater than 97%, equal to or greater than98% or equal to greater than 99%. The position of interest is then giventhe number assigned in the reference nucleic acid molecule. Alignmentcan be effected manually or by eye, particularly, where sequenceidentity is greater than 80%.

As used herein, a human protein is one encoded by a nucleic acidmolecule, such as DNA, present in the genome of a human, including allallelic variants and conservative variations thereof. A variant ormodification of a protein is a human protein if the modification isbased on the wildtype or prominent sequence of a human protein.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, modification is in reference to modification of asequence of amino acids of a polypeptide or a sequence of nucleotides ina nucleic acid molecule and includes deletions, insertions, andreplacements (e.g. substitutions) of amino acids and nucleotides,respectively. Exemplary of modifications are amino acid substitutions.An amino-acid substituted polypeptide can exhibit 65%, 70%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity toa polypeptide not containing the amino acid substitutions. Amino acidsubstitutions can be conservative or non-conservative. Generally, anymodification to a polypeptide retains an activity of the polypeptide.Methods of modifying a polypeptide are routine to those of skill in theart, such as by using recombinant DNA methodologies.

As used herein, suitable conservative substitutions of amino acids areknown to those of skill in this art and can be made generally withoutaltering the biological activity of the resulting molecule. Those ofskill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/CummingsPub. co., p. 224). Such substitutions can be made in accordance withthose set forth in TABLE 2 as follows:

TABLE 2 Exemplary conservative Original residue substitution Ala (A)Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E)Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; ValLys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Leu; Tyr Ser(S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; LeuOther substitutions also are permissible and can be determinedempirically or in accord with known conservative substitutions.

As used herein, the term promoter means a portion of a gene containingDNA sequences that provide for the binding of RNA polymerase andinitiation of transcription. Promoter sequences are commonly, but notalways, found in the 5′ non-coding region of genes.

As used herein, isolated or purified polypeptide or protein orbiologically-active portion thereof is substantially free of cellularmaterial or other contaminating proteins from the cell or tissue fromwhich the protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. Preparationscan be determined to be substantially free if they appear free ofreadily detectable impurities as determined by standard methods ofanalysis, such as thin layer chromatography (TLC), gel electrophoresisand high performance liquid chromatography (HPLC), used by those ofskill in the art to assess such purity, or sufficiently pure such thatfurther purification would not detectably alter the physical andchemical properties, such as enzymatic and biological activities, of thesubstance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound, however, can be amixture of stereoisomers. In such instances, further purification mightincrease the specific activity of the compound.

Hence, reference to a substantially purified polypeptide, refers topreparations of proteins that are substantially free of cellularmaterial including preparations of proteins in which the protein isseparated from cellular components of the cells from which it isisolated or recombinantly-produced. In one embodiment, the termsubstantially free of cellular material includes preparations of enzymeproteins having less than about 30% (by dry weight) of non-enzymeproteins (also referred to herein as a contaminating protein), generallyless than about 20% of non-enzyme proteins or 10% of non-enzyme proteinsor less than about 5% of non-enzyme proteins. When the enzyme protein isrecombinantly produced, it also is substantially free of culture medium,i.e., culture medium represents less than about or at 20%, 10% or 5% ofthe volume of the enzyme protein preparation.

As used herein, the term substantially free of chemical precursors orother chemicals includes preparations of enzyme proteins in which theprotein is separated from chemical precursors or other chemicals thatare involved in the synthesis of the protein. The term includespreparations of enzyme proteins having less than about 30% (by dryweight), 20%, 10%, 5% or less of chemical precursors or non-enzymechemicals or components.

As used herein, synthetic, with reference to, for example, a syntheticnucleic acid molecule or a synthetic gene or a synthetic peptide refersto a nucleic acid molecule or polypeptide molecule that is produced byrecombinant methods and/or by chemical synthesis methods.

As used herein, production by recombinant means or using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, an expression vector includes vectors capable ofexpressing DNA that is operatively linked with regulatory sequences,such as promoter regions, that are capable of effecting expression ofsuch DNA fragments. Such additional segments can include promoter andterminator sequences, and optionally can include one or more origins ofreplication, one or more selectable markers, an enhancer, apolyadenylation signal. Expression vectors are generally derived fromplasmid or viral DNA, or can contain elements of both. Thus, anexpression vector refers to a recombinant DNA or RNA construct, such asa plasmid, a phage, recombinant virus or other vector that, uponintroduction into an appropriate host cell, results in expression of thecloned DNA. Appropriate expression vectors are well known to those ofskill in the art and include those that are replicable in eukaryoticcells and/or prokaryotic cells and those that remain episomal or thosewhich integrate into the host cell genome.

As used herein the term “assessing” is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of a protein, such as an enzyme, or a domainthereof, present in the sample, and also of obtaining an index, ratio,percentage, visual or other value indicative of the level of theactivity. Assessment can be direct or indirect. For example, thechemical species actually detected need not of course be theenzymatically cleaved product itself but can for example be a derivativethereof or some further substance. For example, detection of a cleavageproduct can be a detectable moiety such as a fluorescent moiety.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities can be observed in in vitro systems designed to test or usesuch activities.

As used herein equivalent, when referring to two sequences of nucleicacids, means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with only aminoacid substitutions that do not substantially alter the activity orfunction of the protein or peptide. When equivalent refers to aproperty, the property does not need to be present to the same extent(e.g., two peptides can exhibit different rates of the same type ofenzymatic activity), but the activities are usually substantially thesame.

As used herein, “modulate” and “modulation” or “alter” refer to a changeof an activity of a molecule, such as a protein. Exemplary activitiesinclude, but are not limited to, biological activities, such as signaltransduction. Modulation can include an increase in the activity (i.e.,up-regulation or agonist activity), a decrease in activity (i.e.,down-regulation or inhibition) or any other alteration in an activity(such as a change in periodicity, frequency, duration, kinetics or otherparameter). Modulation can be context dependent and typically modulationis compared to a designated state, for example, the wildtype protein,the protein in a constitutive state, or the protein as expressed in adesignated cell type or condition.

As used herein, a composition refers to any mixture. It can be asolution, suspension, liquid, powder, paste, aqueous, non-aqueous or anycombination thereof.

As used herein, a combination refers to any association between or amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof. The elements of a combination are generally functionallyassociated or related.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from cause or condition including, but notlimited to, infections, acquired conditions, genetic conditions, andcharacterized by identifiable symptoms.

As used herein, treatment means any manner in which the symptoms of acondition, disorder or disease or other indication, are ameliorated orotherwise beneficially altered.

As used herein, a disease or disorder refers to a pathological conditionin an organism resulting from, for example, infection or genetic defect,and characterized by identifiable symptoms. An exemplary disease asdescribed herein is a neoplastic disease, such as cancer.

As used herein, neoplastic disease refers to any disorder involvingcancer, including tumor development, growth, metastasis and progression.

As used herein, cancer is a term for diseases caused by or characterizedby any type of malignant tumor, including metastatic cancers, lymphatictumors, and blood cancers. Exemplary cancers include, but are notlimited to, leukemia, lymphoma, pancreatic cancer, lung cancer, ovariancancer, breast cancer, cervical cancer, bladder cancer, prostate cancer,glioma tumors, adenocarcinomas, liver cancer and skin cancer. Exemplarycancers in humans include a bladder tumor, breast tumor, prostate tumor,basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer,brain and CNS cancer (e.g., glioma tumor), cervical cancer,choriocarcinoma, colon and rectum cancer, connective tissue cancer,cancer of the digestive system; endometrial cancer, esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer;intra-epithelial neoplasm; kidney cancer; larynx cancer; leukemia; livercancer; lung cancer (e.g., small cell and non-small cell); lymphomaincluding Hodgkin's and Non-Hodgkin's lymphoma; melanoma; myeloma,neuroblastoma, oral cavity cancer (e.g., lip, tongue, mouth, andpharynx); ovarian cancer; pancreatic cancer, retinoblastoma;rhabdomyosarcoma; rectal cancer, renal cancer, cancer of the respiratorysystem; sarcoma, skin cancer; stomach cancer, testicular cancer, thyroidcancer; uterine cancer, cancer of the urinary system, as well as othercarcinomas and sarcomas. Exemplary cancers commonly diagnosed in dogs,cats, and other pets include, but are not limited to, lymphosarcoma,osteosarcoma, mammary tumors, mastocytoma, brain tumor, melanoma,adenosquamous carcinoma, carcinoid lung tumor, bronchial gland tumor,bronchiolar adenocarcinoma, fibroma, myxochondroma, pulmonary sarcoma,neurosarcoma, osteoma, papilloma, retinoblastoma, Ewing's sarcoma,Wilm's tumor, Burkitt's lymphoma, microglioma, neuroblastoma,osteoclastoma, oral neoplasia, fibrosarcoma, osteosarcoma andrhabdomyosarcoma, genital squamous cell carcinoma, transmissiblevenereal tumor, testicular tumor, seminoma, Sertoli cell tumor,hemangiopericytoma, histiocytoma, chloroma (e.g., granulocytic sarcoma),corneal papilloma, corneal squamous cell carcinoma, hemangiosarcoma,pleural mesothelioma, basal cell tumor, thymoma, stomach tumor, adrenalgland carcinoma, oral papillomatosis, hemangioendothelioma andcystadenoma, follicular lymphoma, intestinal lymphosarcoma, fibrosarcomaand pulmonary squamous cell carcinoma. Exemplary cancers diagnosed inrodents, such as a ferret, include, but are not limited to, insulinoma,lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALTlymphoma and gastric adenocarcinoma. Exemplary neoplasias affectingagricultural livestock include, but are not limited to, leukemia,hemangiopericytoma and bovine ocular neoplasia (in cattle); preputialfibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma,connective tissue neoplasia and mastocytoma (in horses); hepatocellularcarcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep);pulmonary sarcoma, lymphoma, Rous sarcoma, reticulo-endotheliosis,fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (inavian species); retinoblastoma, hepatic neoplasia, lymphosarcoma(lymphoblastic lymphoma), plasmacytoid leukemia and swimbladder sarcoma(in fish), caseous lymphadenitis (CLA): chronic, infectious, contagiousdisease of sheep and goats caused by the bacterium Corynebacteriumpseudotuberculosis, and contagious lung tumor of sheep caused byjaagsiekte.

As used herein, therapeutic agents are agents that ameliorate thesymptoms of a disease or disorder or ameliorate the disease or disorder.Therapeutic agent, therapeutic compound, or therapeutic regimens includeconventional drugs and drug therapies, including vaccines for treatmentor prevention (i.e., reducing the risk of getting a particular diseaseor disorder), which are known to those skilled in the art and describedelsewhere herein. Therapeutic agents for the treatment of neoplasticdisease or cancer therapy include, but are not limited to, moieties thatinhibit cell growth or promote cell death, that can be activated toinhibit cell growth or promote cell death, or that activate anotheragent to inhibit cell growth or promote cell death. Therapeutic agentsfor use in the methods provided herein can be, for example, ananticancer agent. Exemplary therapeutic agents include, for example,therapeutic microorganisms, such as therapeutic viruses and bacteria,cytokines, growth factors, photosensitizing agents, radionuclides,toxins, antimetabolites, signaling modulators, anticancer antibiotics,anticancer antibodies, angiogenesis inhibitors, radiation therapy,chemotherapeutic compounds or a combination thereof.

As used herein, a “metastasis” refers to the spread of cancer from onepart of the body to another. For example, in the metastatic process,malignant cells can spread from the site of the primary tumor in whichthe malignant cells arose and move into lymphatic and blood vessels,which transport the cells to normal tissues elsewhere in an organismwhere the cells continue to proliferate. A tumor formed by cells thathave spread by metastasis is called a “metastatic tumor,” a “secondarytumor” or a “metastasis.”

As used herein, treatment of a subject that has a neoplastic disease,including a tumor or metastasis, means any manner of treatment in whichthe symptoms of having the neoplastic disease are ameliorated orotherwise beneficially altered. Typically, treatment of a tumor ormetastasis in a subject encompasses any manner of treatment that resultsin slowing of tumor growth, lysis of tumor cells, reduction in the sizeof the tumor, prevention of new tumor growth, or prevention ofmetastasis of a primary tumor, including inhibition vascularization ofthe tumor, tumor cell division, tumor cell migration or degradation ofthe basement membrane or extracellular matrix.

As used herein, the term “wound” refers to a physical trauma to anorganism that can damage cells, tissues, organs and systems of theorganism. Wounds include open wounds, such as incisions, burns,lacerations, abrasions, puncture wounds and penetration wounds, whichare exposed to the environment, and closed wounds, which are typicallyinternal to the organism and include, for example, contusions, hematomasand crushing injuries.

As used herein, inflamed tissue refers to tissue affected byinflammation and affected cells contained within the tissue. The terminflammation is intended to represent the normal response of the immunesystem to infection or irritation.

As used herein, therapeutic effect means an effect resulting fromtreatment of a subject that alters, typically improves or amelioratesthe symptoms of a disease or condition or that cures a disease orcondition. A therapeutically effective amount refers to the amount of acomposition, molecule or compound which results in a therapeutic effectfollowing administration to a subject.

As used herein, amelioration or alleviation of the symptoms of aparticular disorder, such as by administration of a particularpharmaceutical composition, refers to any lessening, whether permanentor temporary, lasting or transient that can be attributed to orassociated with administration of the composition.

As used herein, the term “therapeutic virus” refers to a virus that isadministered for the treatment of a disease or disorder, such as aneoplastic disease, such as cancer, a tumor and/or a metastasis orinflammation or wound or diagnosis thereof and or both. Generally, atherapeutic virus herein is one that exhibits anti-tumor activity andminimal toxicity.

As used herein, a tumor, also known as a neoplasm, is an abnormal massof tissue that results when cells proliferate at an abnormally highrate. Tumors can show partial or total lack of structural organizationand functional coordination with normal tissue. Tumors can be benign(not cancerous), or malignant (cancerous). As used herein, a tumor isintended to encompass hematopoietic tumors as well as solid tumors.

Malignant tumors can be broadly classified into three major types.Carcinomas are malignant tumors arising from epithelial structures (e.g.breast, prostate, lung, colon, pancreas). Sarcomas are malignant tumorsthat originate from connective tissues, or mesenchymal cells, such asmuscle, cartilage, fat or bone. Leukemias and lymphomas are malignanttumors affecting hematopoietic structures (structures pertaining to theformation of blood cells) including components of the immune system.Other malignant tumors include, but are not limited to, tumors of thenervous system (e.g. neurofibromatomas), germ cell tumors, and blastictumors.

As used herein, proliferative disorders include any disorders involvingabnormal proliferation of cells (i.e. cells proliferate more rapidlycompared to normal tissue growth), such as, but not limited to,neoplastic diseases.

As used herein, a “tumor cell” is any cell that is part of a tumor.Typically, the viruses provided herein preferentially infect tumor cellsin a subject compared to normal cells.

As used herein, a “metastatic cell” is a cell that has the potential formetastasis. Metastatic cells have the ability to metastasize from afirst tumor in a subject and can colonize tissue at a different site inthe subject to form a second tumor at the site.

As used herein, “tumorigenic cell,” is a cell that, when introduced intoa suitable site in a subject, can form a tumor. The cell can benon-metastatic or metastatic.

As used herein, a “normal cell” is a cell that is not derived from atumor.

As used herein, the term “cell” refers to the basic unit of structureand function of a living organism as is commonly understood in thebiological sciences. A cell can be a unicellular organism that isself-sufficient and that can exist as a functional whole independentlyof other cells. A cell also can be one that, when not isolated from theenvironment in which it occurs in nature, is part of a multicellularorganism made up of more than one type of cell. Such a cell, which canbe thought of as a “non-organism” or “non-organismal” cell, generally isspecialized in that it performs only a subset of the functions performedby the multicellular organism as whole. Thus, this type of cell is not aunicellular organism. Such a cell can be a prokaryotic or eukaryoticcell, including animal cells, such as mammalian cells, human cells andnon-human animal cells or non-human mammalian cells. Animal cellsinclude any cell of animal origin that can be found in an animal. Thus,animal cells include, for example, cells that make up the variousorgans, tissues and systems of an animal.

As used herein an “isolated cell” is a cell that exists in vitro and isseparate from the organism from which it was originally derived.

As used herein, a “cell line” is a population of cells derived from aprimary cell that is capable of stable growth in vitro for manygenerations. Cell lines are commonly referred to as “immortalized” celllines to describe their ability to continuously propagate in vitro.

As used herein a “tumor cell line” is a population of cells that isinitially derived from a tumor. Such cells typically have undergone somechange in vivo such that they theoretically have indefinite growth inculture; unlike primary cells, which can be cultured only for a finiteperiod of time. Such cells can form tumors after they are injected intosusceptible animals.

As used herein, a “primary cell” is a cell that has been isolated from asubject.

As used herein, a “host cell” or “target cell” are used interchangeablyto mean a cell that can be infected by a virus.

As used herein, the term “tissue” refers to a group, collection oraggregate of similar cells generally acting to perform a specificfunction within an organism.

As used herein, the terms immunoprivileged cells and immunoprivilegedtissues refer to cells and tissues, such as solid tumors, which aresequestered from the immune system. Generally, administration of a virusto a subject elicits an immune response that clears the virus from thesubject. Immunoprivileged sites, however, are shielded or sequesteredfrom the immune response, permitting the virus to survive and generallyto replicate. Immunoprivileged tissues include proliferating tissues,such as tumor tissues.

As used herein, cell therapy refers to treatment with a cell, such as acell transplant. The cell transplant can be selected from among, but notlimited to, pancreatic islet, bone marrow, endothelial, epidermal,myoblast, neural and stem cell transplants. The cell can be modified toexpress a reporter, or can be infected ex vivo, with a virus or viralvector or other eukaryotic vector, to express the reporter. In someembodiments, the cell contains a viral vector, mammalian vector,bacterial vector, insect vector, plant vector or artificial chromosomeencoding a reporter protein. In other embodiments, the cell is infectedwith a virus prior to administration to the subject. In such examples,the virus contains a reporter gene that encodes a reporter protein.

As used herein, a compound produced in a tumor or other immunoprivilegedsite refers to any compound that is produced in the tumor or tumorenvironment by virtue of the presence of an introduced virus, generallya recombinant virus, expressing one or more gene products. For example,a compound produced in a tumor can be, for example, an encodedpolypeptide or RNA, a metabolite, or compound that is generated by arecombinant polypeptide and the cellular machinery of the tumor orimmunoprivileged tissue or cells.

As used herein, a subject includes any organism, including an animal forwhom diagnosis, screening, monitoring or treatment is contemplated.Animals include mammals such as primates and domesticated animals. Anexemplary primate is human. A patient refers to a subject, such as amammal, primate, human, or livestock subject afflicted with a diseasecondition or for which a disease condition is to be determined or riskof a disease condition is to be determined.

As used herein, a delivery vehicle for administration refers to alipid-based or other polymer-based composition, such as liposome,PEGylated liposome, nanoparticle, lipid-based nanoparticle, lymphocyte,micelle or reverse micelle, that associates with an agent, such as avirus provided herein, for delivery into a host subject.

As used herein, a patient refers to a human subject exhibiting symptomsof a disease or disorder.

As used herein, about the same means within an amount that one of skillin the art would consider to be the same or to be within an acceptablerange of error. For example, typically, for pharmaceutical compositions,within at least 1%, 2%, 3%, 4%, 5% or 10% is considered about the same.Such amount can vary depending upon the tolerance for variation in theparticular composition by subjects.

As used herein, animal includes any animal, such as, but are not limitedto primates including humans, gorillas and monkeys; rodents, such asmice and rats; fowl, such as chickens; ruminants, such as goats, cows,deer and sheep; pigs and other animals. Non-human animals exclude humansas the contemplated animal.

As used herein, a control refers to a sample that is substantiallyidentical to the test sample, except that it is not treated with a testparameter, or, if it is a plasma sample, it can be from a normalvolunteer not affected with the condition of interest. A control alsocan be an internal control.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to a compound comprising or containing “anextracellular domain” includes compounds with one or a plurality ofextracellular domains.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 bases” means “about 5 bases” and also “5 bases.”

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance does or does not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not. For example, an optionally substitutedgroup means that the group is unsubstituted or is substituted.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem. 11:1726).

B. OVERVIEW OF METHOD

Provided herein are methods for monitoring biological therapies,including detecting whether they have initiated, their progress andtheir effects. All of the therapies require at least to some extent,amplification of the agent or a product thereof. The methods providedherein detect or identify a product that is produced, either anendogenous or heterologous product of the therapeutic, that reflectsthat the therapeutic has reached or colonized a target in the treatedsubject. Thus, if stem cells are administered, the method detects in asample, such as a body fluid sample, that is different from the locus ofthe treatment, expression of a product of the stem cells. The stem cellsor cells for adoptive immunotherapy can be modified to express a proteinor other reporter, and then, the protein or product of the cells isdetected in a sample. For example, for oncolytic viral therapy, virus isadministered. The viruses accumulate in tumor cells, and as therapyproceeds, the viruses express encoded products that, as shown herein,are shed, or released, and can be detected in body fluids, such asserum, and non-tumor tissues. If therapy is proceeding, the oncolyticviruses replicate and the products can be detected. If therapy is noteffective, then product will not begin to accumulate or be detectable innon-target tissues and body fluids.

Hence, for example, the methods herein, can monitor or detect theefficacy of the biological therapy, for example, by non-invasivelysampling a body fluid and detecting a product, either directly or byvirtue of its activity, that is encoded in the genome of the biologicaltherapy. In some embodiments, the biological therapeutic can be modifiedto express a reporter gene, such as nucleic acid that encodes an enzyme.If therapy is effective, i.e., the stem cells are replicating or theoncolytic virus is accumulating in tumor cells, the activity of thereporter protein, e.g., enzyme, in the sample can be detected, andtypically compared to a control or standard, to show that it isincreased.

In some embodiments of the methods, a subject is treated with abiological therapeutic that encodes a reporter protein. The biologicaltherapeutic can express the reporter constitutively or under the controlof a constitutive promoter or other regulatory signal so thatexpression, for example, only is effected when monitoring is desired.The reporter gene can be introduced into the genome of the biologicaltherapeutic, or can be introduced in a vector. Thus, where the therapyis a cell, such as a stem cell, the stem cell can be modified to expressa reporter, or can be infected ex vivo, with a virus or viral vector orother eukaryotic vector, to express the reporter. Alternatively, asnoted, the reporter can be a protein endogenously encoded. Similarly,for therapies such as oncolytic therapies, in which a virus or bacteriumis administered, the virus or bacterium can be modified to encode thereporter. Reporters include, but are not limited, to enzymes. In someexamples, the vector encodes additional heterologous proteins. Forexample, the vector encodes the reporter gene and an additionalheterologous gene, such as a therapeutic protein. Following or during orintermittently or periodically during treatment with the biologicaltherapeutic, a sample, from a locus other than the target tissue or cellof the therapy, such as blood, serum, saliva, and urine, is collectedfrom the subject. If the reporter is an enzyme, the sample or portionthereof, is contacted with a suitable substrate. Following incubation ofthe sample and the substrate, the sample is monitored to detect thereporter protein or a signal induced by the reporter protein. Detectionof the reporter protein allows for in vivo monitoring of viral therapy,bacterial therapy, cell therapy, immunotherapy, adoptive immunotherapyor gene therapy.

1. Biological Therapies

The use of biological therapies, such as stem cell therapy, genetherapy, immunotherapy, oncolytic virotherapy, oncolytic bacterialtherapy, cell therapy for regenerative medicine, immunology, oncologyand the treatment of various diseases is growing. A feature of thesetherapies is the required expression of genes that usually are not orare only weakly expressed in the targeted tissue; or, for oncolytictherapies, replication of the virus or bacterium or other oncolyticvector is required. As shown herein, if the therapy has achieved aneffect, such as colonization of target tissues, or replication of anoncolytic virus, it is possible to detect products encoded by thebiological therapeutic in loci distinct from the target of thetherapeutic. These loci include body fluids and tissues thatconveniently can be sampled and tested for a product encoded by thetherapeutic. The encoded product can be one not normally expressed inthe subject in which case detection indicates that the therapeutic isreplicating or expressing product, or the product can be one that isproduced in which case an increase compared to a standard or thesubject's baseline level or other suitable control is assessed. Thefollowing discussion describes exemplary biological therapeutics. Theseare exemplary and not to be construed to be limiting of those for whichtreatment can be monitored by the methods herein.

a. Oncolytic Viral Therapy

Numerous oncolytic viruses have been identified or developed. Theseinclude vaccinia viruses, vesticular stomatis viruses and adenoviruses.When administered, the viruses accumulate in tumor cells and tissues,replicate and lyse the cells, releasing the contents thereof. As shownherein, if the virus is replicating or expressing encoded gene products,these products can be detected in non-tumor tissue, thereby serving as amarker for viral colonization/infection of the tumor cell. The amountexpressed and/or expression can be used to assess effectiveness of thetherapy. Oncolytic viruses have been genetically altered to attenuatetheir virulence, to improve their safety profile, enhance their tumorspecificity, and they have also been equipped with additional genes, forexample cytotoxins, cytokines, or prodrug converting enzymes to improvethe overall efficacy of the viruses (see, e.g., Kirn et al., (2009) NatRev Cancer 9:64-71; Garcia-Aragoncillo et al., (2010) Curr Opin Mol Ther12:403-411; see U.S. Pat. Nos. 7,588,767, 7,588,771, 7,662,398 and7,754,221 and U.S. Pat. Publ. Nos. 2007/0202572, 2007/0212727,2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287, 2009/0117034,2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and 2011/0064650.

Vaccinia viruses, particular those that are modified to have decreasedvirulence or that have decreased virulence, are among those that areparticularly advantageous. For example, vaccinia virus is a large DNAvirus that encodes its own DNA polymerase such that it is able toreplicate in the cytoplasm of infected host cells thereby minimizing therisk of DNA integration into the host genome. Vaccinia virus displays abroad host cell range, rapid spread and a high capacity (up to about 25kbp) for genetic payload of foreign DNA (Moss et al., (1996) Proc NatlAcad Sci USA 93:11341-11348). Of note and importance regarding thesafety of vaccinia virus, is also its billion-fold use in humans duringthe eradication program of smallpox, as well as the fact that vacciniavirus is not a human pathogen. Further, certain vaccinia virus strainshave been shown to specifically colonize solid tumors, while notinfecting other organs (Zhang et al. (2007) Cancer Res 67:10038-10046;Yu et al., (2004) Nat Biotech 22:313-320; Heo et al., (2011) Mol Ther19:1170-1179; Liu et al. (2008) Mol Ther 16:1637-1642; Park et al.,(2008) Lancet Oncol, 9:533-542; Pedersen et al.: Preliminary results ofa Phase 1 study of intravenous administration of GL-ONC1 Vaccinia virusin patients with advanced solid cancer with real time imaging. In 6thNCRI Cancer Conference; BT Convention Center, Liverpool, UK. 2010). Themethods provided herein permit reliable monitoring of successful tumorcolonization in humans. This has an enormous impact, not only onclinical trials, but also for predicting outcomes of oncolytic virustherapy.

In practicing the methods herein, the presence of endogenous and/orheterologous gene products in non-target samples from treated subjectsis assessed as an indicator of the effectiveness or progress of thebiological therapy. Among the gene products that can be detected arevarious reporter genes that have been used for optical (e.g. Puhlmann etal., (2000) Cancer Gene Ther 7:66-73) and radiological (e.g. Bennett etal., (2001) Nat Med 7:859-863; Chen et al., (2009) Mol Med 15:144-151;Dingli et al., (2004) Blood 103:1641-1646; Haddad et al., (2011) JTransl Med 9:36) imaging. The methods herein have advantages compared tooptical and radiological imaging in treated subjects. For example,optical imaging has limitations in penetration depth and radiologicalimaging is time consuming and requires specialized personnel andexpensive equipment.

2. Beta-Glucuronidases as Reporter Proteins

Exemplary of the reporter proteins contemplated herein arebeta-glucuronidases. Beta-glucuronidases (β-glucuronidase, GusA)catalyze the hydrolysis of β-D-glucuronides into the correspondingD-glucuronate and alcohol. Mammalian β-glucuronidases have a pH-optimumunder acidic conditions (pH 4 to 5) and have reduced capacity at normal(neutral) tissue pH. The bacterial enzyme, E. coli β-glucuronidaseencoded by the gene designated gusA has optimal activity in the range ofpH 6.8 to 7.7 (see, e.g., Fang et al., (1995) Vet Microbiol 46:361-367).β-glucuronidase has been used in plant physiology studies (Jefferson etal., (1986) Proc Natl Acad Sci USA 83:8447-8451; and Jefferson et al.,(1987) EMBO J 6:3901-3907). In mammals, bacterial glucuronidase has beenused as a reporter in prodrug studies, due to the very low abundance ofhuman glucuronidase in serum (Stahl and Fishman: Beta-D-glucuronidase.In Methods in enzymatic analysis. Edited by J B, M G. Weinheim, Germany:Verlag Chemie; 1984: 246-256). Several strategies have been employed,including fusion of cancer specific antibody-fragments withbeta-glucuronidase (Wang et al., (1992) Cancer Res 52:4484-4491), andtumor selective expression of the enzyme using bacteria (Cheng et al.,(2008) Cancer Gene Ther 15:393-401) or adenovirus (de Graaf et al.,(2004) Human Gene Ther 15:229-238; Huang et al., (2011) Cancer Gene Ther18:381-389). The reporter gene properties of β-glucuronidase have notbeen studied as extensively in animals. Beta-glucuronidase has beenconsidered as a target structure for radiotracers in positron emissiontomography (Tzou et al., (2009) Radiology 252:754-762; Antunes et al.,(2010) Bioconjug Chem 21:911-920). In another study, a membrane-anchoredform of a mouse-glucuronidase was used in combination with the substratefluorescein di-β-D-glucuronide (FDGlcU), which was hydrolyzed to afluorescent reporter to assess the location and persistence of geneexpression in vivo (Su et al., (2007) Gene Ther 14:565-574).

Bacterial glucuronidase is an advantageous reporter enzyme for methodsin which body fluids such as urine and blood and serum are sampled. Ithas a pH-optimum range of pH 6.8 to 7.7 (Fang et al., (1995) VetMicrobiol 46:361-367), and has higher specific activity than humanbeta-glucuronidase (Chen et al., (2008) Chem Biol 15:1277-1286).Furthermore, the possibility to direct active beta-glucuronidase intothe cytoplasm (see, e.g. Jefferson et al., (1986) Proc Natl Acad Sci USA83:8447-8451), attach it to a cell surface (e.g. Huang et al., (2011)Cancer Gene Ther 18:381-389, Heine et al., (2001) Gene Ther8:1005-1010), or secrete it from producing cells (e.g. Chen et al.,(2011) Bioconjug Chem 22:938-948; Weyel et al., (2000) Gene Ther7:224-231) offers a number of different applications.

In the methods herein, this enzyme, and/or other proteins, is detectedin tissue and fluid samples distinct from the therapeutic target, toassess whether a biological therapeutic has colonized or replicated in atarget tissue that is distinct from the tissue and fluid sample.

3. Method

Methods for detecting colonization and/or infection of target cells andloci by biological therapeutics and for monitoring therapy are provided.As described herein, the methods detect a product of the biologicaltherapeutic in a locus distinct from the target of the therapeutic.Thus, for example, a body fluid, such as serum or urine or CSF orsaliva, can be sampled to detect a product produced by the therapeuticin another locus. Presence of the gene product in untreated tissues andbody fluids or untreated or normal tissue samples indicates that thebiological therapy has colonized or infected the target loci in thetreated subject. As described herein, any reporter or product of thebiological therapeutic can be employed, including endogenous andheterologous reporter gene products and signals. Production of suchproducts and their presence in non-treated tissues, cells and/or bodyfluids is indicative of the effectiveness of the biological therapeuticand progress of treatment. Thus, the effectiveness and progress oftherapy with a biological therapeutic can be monitored.

Reporter gene products, such as enzymes, are exemplary of the geneproducts that can be monitored. As a non-limiting example, the activityof the enzyme beta-glucuronidase is detected by contacting a sample,such as a body fluid or tissue that does not contain treated tissue orcells, from a treated host, with a substrate for the enzyme to produce aproduct that is detected. The detected product is correlated with theprogress or status of the therapy. For exemplary purposes, vacciniavirus (rVACV), particularly an LIVP strain or LIVP, that encodesβ-glucuronidase is employed as a general marker for the preclinical andclinical evaluation of biological therapies. Tumor colonization byoncolytic rVACV results in tumor-specific expression of virus encodedproteins that are shed after successful tumor colonization andreplication. Successful treatment with an oncolytic rVACV that containsa β-glucuronidase gene is assessed by detecting expression ofβ-glucuronidase in non-treated host cells, tissues and/or body fluids,and therefore, detection of virus mediated enzyme activity. As describedherein, enzyme activity was detected in serum samples of solidtumor-bearing subjects that had been treated with rVACV, thereby,non-invasively confirming successful tumor colonization. In addition, atherapeutic effect was observed as active enzyme can only get in theserum when tumor cells are lysed and the protein is released, or shed.

Thus, provided herein is a method for monitoring biological therapies bydetection of reporter protein in non-treated samples or non-treated lociin treated subjects. In the methods, a subject is treated with abiological therapeutic. In some instances, the biological therapeuticencodes a reporter protein and optionally one or more additionalheterologous or exogenous gene products. Any reporter protein can beused in the methods provided herein. In some examples, the reporterprotein is an enzymatic reporter protein. Following or during treatmentwith the biological therapeutic, a sample is collected from the subjectand a product encoded by the therapeutic is detected. If, for example,the product is an enzyme, the sample is contacted with a substrate, andthe resulting product is detected. Following incubation of the sampleand the substrate, the sample is monitored to detect the encodedreporter protein or a signal induced by the reporter protein. Detectionof the reporter protein indicates that the biological therapeutic isreplicating in or colonizing a target locus. Thus, detection ofexpression of proteins introduced by the biological therapy in locidistinct from the treatment's target is indicative of effectivebiological therapy.

In a particular example herein, a substrate that is activated byβ-glucuronidase is added to a biological sample, for example, blood,e.g., serum, or urine, taken from a subject treated, such as by I.V.administration, with any GusA-encoding therapeutic, including, but notlimited to GusA-encoding viral vectors, such as GusA-encoding vacciniavectors, e.g., GusA-rVACV, GusA-expressing bacteria or any otherGusA-encoding vector for heterologous gene expression. After apre-determined reaction time, the sample is analyzed for GusA activatedsubstrate. The method allows for direct diagnosis of viral tumorcolonization in tumor bearing patients.

As exemplified herein, oncolytic vaccinia strains (rVACV) strains thatencode a bacterial beta-glucuronidase were administered to tumor-bearingsubjects. Body fluid samples were obtained and mixed with fluorogenicprobe. As demonstrated herein, the use of fluorogenic probes that arespecifically activated by β-glucuronidase resulted in 1) preferentialactivation in tumors; 2) renal excretion of the activated fluorescentcompounds; and 3) reproducible detection of β-glucuronidase in the serumof oncolytic vaccinia virus treated, tumor bearing mice in several tumormodels. Time course studies demonstrate reliable differentiation betweentumor bearing and healthy mice is possible as early as 9 days postinjection of the virus.

Further, the described method's sensitivity was demonstrated in that asingle infected tumor cell was reliably detected using the assay. Thus,it was determined that as few as or about 2.4×10⁴ vaccinia virusinfected cancer cells are detectable in a human subject using theprovided method. With a diameter of 50 μm/cell, this number correspondsto a tumor volume of only 1.6 mm³. In human subjects, for example, evenif not all virus infected cells release all of their enzyme product intothe blood stream, e.g., serum, upon lysis, such that the number ofinfected cells is 10 or even 100-fold higher than detected, the tumordiameter necessary for positive detection using the methods providedherein is as small as 3.1 and 6.7 mm³, respectively. Thus, the methodsherein can detect tumors as small as about 1-3 mm³, even assuming thatonly a fraction of the infected/colonized tumor cells release product.

For example, serum was obtained from tumor- and non-tumor bearing mice,to which a GusA-rVACV (a recombinant vaccinia virus containing the GusAgene) or controls that were either mock or control-rVACV (vaccinia virusnot encoding GusA) were administered. Subsequently, the serum was usedto assess enzyme activity by GusA-activatable fluorescent compounds(namely FDGlcU and 4-MUG). Both compounds were shown to be specificallyactivated in GusA-rVACV-injected tumor bearing mice but not in controlrVACV-injected mice.

Further, as described herein, using E. coli Nissle 1917 to produceproducts for fluorescent compound activation, e.g., FDGlcU or 4-MUGactivation, fluorescent activation was observed in serum derived fromtumor bearing mice that were injected with E. coli Nissle 1917 xpBR322DESTinv-PS10-gusA-1uxABCDE (-SgusAL) but not in serum derived frommice that were either mock or E. coli Nissle 1917 xpBR322DESTinv-PS10-1uxABCDE-term (-SLT) injected mice.

Thus, it has been demonstrated that a reporter product, suchbeta-glucuronidase in combination with fluorogenic substrates cannotonly be used for localization of enzyme expression, but also as ageneral biomarker for foreign protein expression in serum samples.Consequently, the described method can be applied to any biologicaltherapeutic.

C. METHODS FOR DETECTING REPLICATION OR COLONIZATION OF A BIOLOGICALTHERAPEUTIC

Provided herein is a method for detecting replication or colonization ofa biological therapy or biological therapeutic. Biological therapiesinclude, but are not limited to, cell therapy, gene therapy,immunotherapy, adoptive immunotherapy, viral therapy and bacterialtherapy. Thus, the provided method can be used to determine geneexpression of a gene encoded by the therapeutic and/or tumorcolonization of an immunoprivileged cell. Hence, the method providedherein can be used, for example, to determine the presence of a tumor ortumor cells, such as circulating tumor cells and metastasizing cells,tumor colonization by an oncolytic viral therapy, or expression of agene introduced by gene therapy or cell therapy.

Provided herein is a method for detecting replication or colonization ofa target locus in a subject by a biological therapeutic. A sample isobtained from a subject to whom the therapeutic is administered from alocus in the subject other than a target of the biological therapeuticand the sample is tested to detect a protein encoded by the biologicaltherapeutic. Detection of the protein indicates that the biologicaltherapeutic is replicating or colonizing the target locus. The proteinencoded by the biological therapeutic appears in biological tissues andfluids because of replication and colonization by the biologicaltherapeutic, that is, the protein is shed. The provided methodinvolves: 1) treating a subject with a biological therapeutic; 2)collecting a sample from the subject to whom the biological therapeuticwas administered from a locus other than the target locus or collectinga sample that is not targeted tissue; and 3) detecting the presence of aprotein encoded by the biological therapeutic in the sample, therebydetermining the status or progress of therapy by the biologicaltherapeutic. If, for example, the biological therapeutic encodes anenzyme, a substrate for the enzyme is added to the sample underconditions appropriate for enzymatic activity, and the convertedsubstrate (product) is detected either by its detection or detecting asignal produced by the reaction of the enzyme and substrate. Detectionof the signal or protein product is indicative of gene expression of aprotein encoded by the therapeutic, thereby indicating infection of acell or colonization of the subject by the biological therapeutic. Forexample, if the therapeutic is an oncolytic virus, detection of aviral-encoded protein in a body fluid sample is indicative of infectionof tumor cells, including circulating tumor cells (or other cells inwhich such viruses accumulate).

Treatment with the biological therapeutic can be monitored once orperiodically or intermittently and before, during or after treatment.Monitoring the production of such protein during treatment is indicativeof the progress of therapy. Therapy can be monitored over time toindicate the progress of the therapy. For example, the biologicaltherapeutic can be monitored shortly after treatment, wherein enzymedetection indicates viral replication. Over time, the treatment with thebiological therapeutic can be monitored to detect, for example, tumorshrinkage, wherein less enzyme is detected as the tumor volumedecreases, due to a decrease in shed reporter enzyme.

The biological therapeutic can be administered by any method known toone of skill in the art. In one example, the biological therapeutic canbe administered systemically. In another example, the biologicaltherapeutic is administered topically, locally, enterally orparenterally. For example, the biological therapeutic is administeredtopically, such as epicutaneously (onto the skin) or intranasally,enterally, such as orally, by gastric feeding tube, duodenal feedingtube, or gastrostomy, or rectally, parenterally, such as intravenously,intraarterially, intramuscularly, intracardiacly, subcutaneously, byintraosseous infusion (into the bone marrow), intradermally,intraperitoneally, intrapleurally, transdermally, transmucosally, or byother administration including epiduralally, intrathecally,intraventricularly or intratumorally.

Also provided herein is a method for rapidly diagnosing bacteriainvolved in infection, for example, bacteria involved in sepsis andbladder or urinary tract infections. In the provided method,β-glucuronidase is detected in a sample from a subject suspected ofhaving an infection. A sample is obtained, a substrate forβ-glucuronidase is added, and a signal induced by the β-glucuronidase isdetected. Positive detection of β-glucuronidase activity indicates anEscherichia, Shigella and Salmonella infection due to the fact thatβ-glucuronidase activity only occurs in bacteria of the generaEscherichia, Shigella and Salmonella (Kilian and Bulow (1976) ActaPathol Microbiol Scand B. 84B(5):245-251). Thus, using the describedmethod, one can analyze, for example, blood or urine samples using aβ-glucuronidase substrate and rapidly determine if the infection is fromE. coli, for example, as opposed to a different bacteria, such asStaphylococcus. Positive detection of an infection allows treatment withan antibiotic appropriate specific to the identified infection beforethe bacterial strain can be identified via PCR (currently in clinicaltests) or culture.

The following sections describe exemplary biological therapeutics thatcan be monitored using the methods provided herein, including viruses,bacteria, cell therapies and gene therapies. Also described are reporterproteins and substrates, samples and detection methods for use inpracticing the methods.

1. Biological Therapeutics

The methods provided herein allow for detection of replication orcolonization of a biological therapeutic in a subject. Any biologicaltherapeutic can be used in the methods provided. In some examples, thebiological therapeutic contains a vector that encodes or is modified toencode a reporter protein. The vector can be an eukaryotic orprokaryotic vector, such as a viral vector, mammalian vector, bacterialvector, plant vector or insect vector. In some examples, the reporterprotein is encoded by an artificial chromosome. Typically, thebiological therapeutic is a virus or viral vector, bacterium, genetherapy vector or nucleic acid for gene therapy, cells for immunotherapyand/or adoptive immunotherapy, autologous cell therapy or cell therapy.Thus, biological therapeutics for use in the methods provided hereininclude, for example, viruses, e.g. oncolytic viruses, bacteria, e.g. E.coli strain Nissle 1917, gene therapy, immunotherapy or adoptiveimmunotherapy, and cell therapy. The biological therapeutics providedherein can be used to treat diseases and disorders, such as cancer,genetic disorders, and immune disorders.

a. Viruses

The biological therapy for use in the methods provided herein can be avirus, typically an oncolytic virus for use in oncolytic viral therapy.While the protein for detection can be an endogenous protein, theviruses optionally contain heterologous nucleic acid encoding a reporterprotein, e.g., an enzyme, and optionally additionally contain one ormore additional heterologous nucleic acid sequences for the expressionof additional heterologous genes. The heterologous nucleic acid istypically operably linked to a regulatory sequences, including asuitable promoter for expression of the heterologous nucleic acid in theinfected cells. Expression can be constitutive or inducible.

Viruses whose therapies can be monitored include oncolytic viruses,including viruses that accumulate in tumor cells and viruses modified todo so, and viruses for delivery of gene therapy products either byaccumulation in target cells or provided in the infected cell. Virusesand viral vectors include, but are not limited to, poxviruses,herpesviruses, adenoviruses, adeno-associated viruses, lentiviruses,retroviruses, rhabdoviruses, papillomaviruses, vesicular stomatitisvirus, measles virus, Newcastle disease virus, picornavirus, sindbisvirus, papillomavirus, parvovirus, reovirus, coxsackievirus, influenzavirus, mumps virus, poliovirus, and semliki forest virus. Typically, thevirus is a cytoplasmic virus which does not require entry of viralnucleic acid molecules into the nucleus of the host cell during theviral life cycle. A variety of cytoplasmic viruses are known, including,but not limited to, poxviruses, African swine flu family viruses, andvarious RNA viruses such as picornaviruses, caliciviruses, togaviruses,coronaviruses and rhabdoviruses. Exemplary cytoplasmic viruses providedherein are viruses of the poxvirus family, including orthopoxviruses.Exemplary of poxviruses are vaccinia viruses.

i. Poxviruses

In some examples, the therapeutic virus is from the poxvirus family.Poxviruses include Chordopoxviridae, such as orthopoxvirus,parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,molluscipoxvirus and yatapoxvirus, as well as Entomopoxvirinae such asentomopoxvirus A, entomopoxvirus B, and entomopoxvirus C. One skilled inthe art can select a particular genera or individual chordopoxviridaeaccording to the known properties of the genera or individual virus, andaccording to the selected characteristics of the virus (e.g.,pathogenicity, ability to elicit an immune response, preferential tumorlocalization), the intended use of the virus, the tumor type and thehost organism. Exemplary chordopoxviridae genera are orthopoxvirus andavipoxvirus. Avipoxviruses infect a variety of different birds and havebeen administered to humans. Exemplary avipoxviruses include canarypox,fowlpox, juncopox, mynahpox, pigeonpox, psittacinepox, quailpox,peacockpox, penguinpox, sparrowpox, starlingpox, and turkeypox viruses.

Orthopoxviruses infect a variety of different mammals including rodents,domesticated animals, primates and humans. Several orthopoxviruses havea broad host range, while others have narrower host range. Exemplaryorthopoxviruses include buffalopox, camelpox, cowpox, ectromelia,monkeypox, raccoon pox, skunk pox, tatera pox, uasin gishu, vaccinia,variola, and volepox viruses. In some embodiments, the orthopoxvirusselected can be an orthopoxvirus known to infect humans, such as cowpox,monkeypox, vaccinia, or variola virus. Optionally, the orthopoxvirusknown to infect humans can be selected from among orthopoxviruses with abroad host range, such as cowpox, monkeypox, and vaccinia virus.

(1) Vaccinia Viruses

One exemplary orthopoxvirus for therapy that can be monitored anddetected by the methods provided herein is vaccinia virus. Vaccinia is acytoplasmic virus, thus, it does not insert its genome into the hostgenome during its life cycle. The linear dsDNA viral genome of vacciniavirus is approximately 200 kb in size, encoding a total of approximately200 genes. A variety of vaccinia virus strains are available for therapyand/or diagnostics. These include, but are not limited to strains of orderived from, Western Reserve (WR) (SEQ ID NO:104), Copenhagen (SEQIDNO:105), Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W,Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8, LC16MO, LIVP, WR 65-16,Connaught, New York City Board of Health strains. Exemplary vacciniaviruses are Lister, particularly LIVP, vaccinia viruses. In oneembodiment, the Lister strain can be an attenuated Lister strain, suchas the LIVP (Lister virus from the Institute of Viral Preparations,Moscow, Russia) strain, which was produced by further attenuation of theLister strain. The LIVP strain was used for vaccination throughout theworld, particularly in India and Russia, and is widely available. Inanother embodiment, the viruses and methods provided herein can be basedon modifications to the Lister strain of vaccinia virus.

Lister (also referred to as Elstree) vaccinia virus is available fromany of a variety of sources. For example, the Elstree vaccinia virus isavailable at the ATCC under Accession Number VR-1549. The Listervaccinia strain has high transduction efficiency in tumor cells withhigh levels of gene expression. LIVP and its production are described inU.S. Pat. Nos. 7,588,767, 7,588,771, 7,662,398 and 7,754,221 and U.S.Patent Publication Nos. 2007/0202572, 2007/0212727, 2010/0062016,2009/0098529, 2009/0053244, 2009/0155287, 2009/0117034, 2010/0233078,2009/0162288, 2010/0196325, 2009/0136917 and 2011/0064650 and SEQ IDNOS:82-91.

Vaccinia virus, particularly strains modified or identified as havingreduced toxicity, possesses a variety of features advantageous in cancergene therapy and vaccination including broad host and cell type range, alarge carrying capacity for foreign genes (up to 25 kb of exogenous DNAfragments (approximately 12% of the vaccinia genome size) can beinserted into the vaccinia genome), high sequence homology amongdifferent strains for designing and generating modified viruses in otherstrains, and techniques for production of modified vaccinia strains bygenetic engineering are well established (Moss (1993) Curr. Opin. Genet.Dev. 3: 86-90; Broder and Earl (1999) Mol. Biotechnol. 13: 223-245;Timiryasova et al. (2001) Biotechniques 31: 534-540). A variety ofvaccinia virus strains are available, including Western Reserve (WR),Copenhagen, Tashkent, Tian Tan, Lister, Wyeth, IHD-J, and IHD-W,Brighton, Ankara, MVA, Dairen I, LIPV, LC16M8, LC 16Mo, LIVP, WR 65-16,Connaught, New York City Board of Health. Exemplary therapeutic vacciniaviruses include, but are not limited to, Lister strain or LIVP strain ofvaccinia viruses.

LIVP strains that can be used in the methods provided herein includeLIVP clonal strains derived from LIVP that have a genome that is or isderived from or is related to a the parental sequence set forth in SEQID NO:91 (see U.S. patent application Ser. No. 13/506,369 which isincorporated herein by reference). These include the strain designedGLV-1h68 and all strains and modified forms thereof (see, e.g., U.S.Pat. Nos. 7,588,767, 7,588,771, 7,662,398, 7,754,221, 8,021,662,8,052,962 and 8,066,984 and U.S. Patent Publication Nos. 2007/0202572,2007/0212727, 2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287,2009/0117034, 2010/0233078, 2009/0162288, 2010/0196325, 2009/0136917 and2011/0064650). These also include LIVP clonal strains that have beenshown to exhibit greater anti-tumorigenicity and/or reduced toxicitycompared to the recombinant or modified virus strain designated GLV-1h68(having a genome set forth in SEQ ID NO:90; and U.S. patent applicationSer. No. 13/506,369). In particular, the clonal strains are present in avirus preparation propagated from LIVP.

The LIVP and clonal strains for use in the methods provided herein havea sequence of nucleotides that have at least 70%, such as at least 75%,80%, 85% or 90% sequence identity to SEQ ID NO:91. For example, theclonal strains have a sequence of nucleotides that is at or at least91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical to SEQ ID NO:91.Such LIVP clonal viruses include viruses that differ in one or more openreading frames (ORF) compared to the parental LIVP strain that has asequence of nucleotides set forth in SEQ ID NO:91. The LIVP clonal virusstrains provided herein can contain a nucleotide deletion or mutation inany one or more nucleotides in any ORF compared to SEQ ID NO:91, or cancontain an addition or insertion of viral DNA compared to SEQ ID NO:91.

(2) Modified Vaccinia Viruses

Exemplary vaccinia viruses for use as therapeutics in the methods hereininclude vaccinia viruses with insertions, mutations or deletions.Exemplary insertions, mutations or deletions include those that resultin an attenuated vaccinia virus relative to the wild type strain. Forexample, vaccinia virus insertions, mutations or deletions can decreasepathogenicity of the vaccinia virus, for example, by reducing thetoxicity, reducing the infectivity, reducing the ability to replicate,or reducing the number of non-tumor organs or tissues to which thevaccinia virus can accumulate. Other exemplary insertions, mutations ordeletions include, but are not limited to, those that increaseantigenicity of the virus, those that permit detection, monitoring, orimaging, those that alter attenuation of the virus, and those that alterinfectivity. For example, the ability of vaccinia viruses providedherein to infect and replicate within tumors can be enhanced bymutations that increase the extracellular enveloped form of the virus(EEV) that is released from the host cell. Modifications can be made,for example, in genes that are involved in nucleotide metabolism, hostinteractions and virus formation or at other nonessential gene loci. Anyof a variety of insertions, mutations or deletions of the vaccinia virusknown in the art can be used herein, including insertions, mutations ordeletions of: the thymidine kinase (TK) gene, the hemagglutinin (HA)gene, and F14.5L gene, among others (e.g., E2L/E3L, K1L/K2L, superoxidedismutase locus, 7.5K, C7-K1L, J2R, B13R+B14R, A56R, A26L or I4L geneloci). The vaccinia viruses for use in the methods provided herein alsocan contain two or more insertions, mutations or deletions. Thus,included are vaccinia viruses containing two or more insertions,mutations or deletions of the loci provided herein or other loci knownin the art. The viruses can be based on modifications to the Listerstrain and/or LIVP strain of vaccinia virus. Any known vaccinia virusmodification, or modifications that correspond to those provided hereinor known to those of skill in the art to reduce toxicity of a vacciniavirus, can be included a modified vaccinia virus used in the methodsherein. Generally, however, the mutation will be a multiple mutant andthe virus will be further selected to reduce toxicity.

The modified viruses for use in the methods provided herein can encodeheterologous gene products. The heterologous nucleic acid is typicallyoperably linked to a promoter for expression of the heterologous gene inthe infected cells. Suitable promoter include viral promoters, such as avaccinia virus natural and synthetic promoters. Exemplary vaccinia viralpromoters include, but are not limited to, P11k, P7.5k early/late, P7.5kearly, P28 late, synthetic early P_(SE), synthetic early/late P_(SEL)and synthetic late P_(SL) promoters.

The viruses can express one or more genes whose products are useful fortumor therapy. For example, a virus can express proteins that cause celldeath or whose products cause an anti-tumor immune response. Such genescan be considered therapeutic genes. A variety of therapeutic geneproducts, such as toxic or apoptotic proteins, or siRNA, are known inthe art, and can be used with the viruses provided for use as biologicaltherapeutics herein. The therapeutic genes can act by directly killingthe host cell, for example, as a channel-forming or other lytic protein,or by triggering apoptosis, or by inhibiting essential cellularprocesses, or by triggering an immune response against the cell, or byinteracting with a compound that has a similar effect, for example, byconverting a less active compound to a cytotoxic compound. Exemplaryproteins useful for tumor therapy include, but are not limited to, tumorsuppressors, toxins, cytostatic proteins, antiangiogenic proteins,antitumor antibodies, and costimulatory molecules, such as cytokines andchemokines among others provided elsewhere herein and known in the art.The viruses used in the methods provided herein also can be effectiveagainst tumors without the introduction of additional exogenoustherapeutic genes.

The viruses used in the methods provided herein can express one or moreadditional genes whose products are useful for tumor detection and/orimaging. Exemplary gene products for imaging or detection includedetectable proteins or proteins that induce detectable signals.Exemplary of detectable proteins or proteins that induce detectablesignals are proteins, such as luciferases, fluorescent proteins,receptors that can bind imaging agents, or proteins linked to imaging ordiagnostic moieties. The viruses used as therapeutic proteins in themethods provided herein also can encode proteins, such as transporterproteins (e.g., the human norepinephrine transporter (hNET) or the humansodium iodide symporter (hNIS)), which can provide increase uptake ofdiagnostic and therapeutic moieties across the cell membrane of infectedcells for therapy, imaging or detection.

Imaging or diagnostic moieties include those that can emit a signal thatis detectable by optical or non-optical imaging methods. Detection ofthe signal by imaging modalities such as, for example, by positronemission tomography (PET) and, thereby allows visualization of theinfected tissues, such as a tumor or an inflammation.

One skilled in the art can select a virus for use as a biologicaltherapeutic herein from any of a variety of viruses, according to avariety of factors, including, but not limited to, the intended use ofthe virus, such as a diagnostic and/or therapeutic use (e.g., tumortherapy or diagnosis, vaccination, antibody production, or heterologousprotein production), the host organism, and the type of tumor. Anoncolytic virus for use in the methods provided herein can exhibit oneor more desired characteristics for use as a therapeutic agent, such as,for example attenuated pathogenicity, reduced toxicity, preferentialaccumulation in immunoprivileged cells and tissues, such as tumor,ability to activate an immune response against tumor cells, immunogenic,replication competent, and ability to express exogenous proteins, andcombinations thereof.

(3) Exemplary Modified Vaccinia Viruses

Exemplary therapeutic vaccinia viruses include those derived fromvaccinia virus strain GLV-1h68 (also named RVGL21, SEQ ID NO:90), whichhas been described in U.S. Pat. Pub. No. 2005-0031643 and U.S. Pat. No.7,588,767 which are incorporated herein by reference in their entirety.GLV-1h68 contains DNA insertions gene loci of the vaccinia virus LIVPstrain (SEQ ID NO: 91, a vaccinia virus strain, originally derived byadapting the Lister strain (ATCC Catalog No. VR-1549) to calf skin(Institute of Viral Preparations, Moscow, Russia, Al'tshtein et al.,(1983) Dokl. Akad. Nauk USSR 285:696-699)). GLV-1h68 contains expressioncassettes encoding detectable marker proteins in the F14.5L (alsodesignated in LIVP as F3), thymidine kinase (TK) and hemagglutinin (HA)gene loci. An expression cassette containing a Ruc-GFP cDNA molecule (afusion of DNA encoding Renilla luciferase and DNA encoding GFP) underthe control of a vaccinia synthetic early/late promoter P_(SEL)((P_(SEL))Ruc-GFP) is inserted into the F14.5L gene locus; an expressioncassette containing a DNA molecule encoding beta-galactosidase under thecontrol of the vaccinia early/late promoter P_(7.5k) ((P_(7.5k))LacZ)and DNA encoding a rat transferrin receptor positioned in the reverseorientation for transcription relative to the vaccinia syntheticearly/late promoter P_(SEL) ((P_(SEL))rTrfR) is inserted into the TKgene locus (the resulting virus does not express transferrin receptorprotein since the DNA molecule encoding the protein is positioned in thereverse orientation for transcription relative to the promoter in thecassette); and an expression cassette containing a DNA molecule encodingβ-glucuronidase under the control of the vaccinia late promoter P_(11k)((P_(11k))gusA) is inserted into the HA gene locus. The GLV-1h68 virusexhibits a strong preference for accumulation in tumor tissues ascompared to non-tumorous tissues following systemic administration ofthe virus to tumor bearing subjects. This preference is significantlyhigher than the tumor selective accumulation of other vaccinia viralstrains, such as WR (see, e.g. U.S. Pat. Pub. No. 2005-0031643 and Zhanget al. (2007) Cancer Res. 67(20):10038-10046). Modified viruses for usein the methods provided herein can be derived from GLV-1h68. Exemplaryviruses are generated by replacement of one or more expression cassettesof the GLV-1h68 strain with heterologous DNA encoding gene products fortherapy and/or imaging.

Non-limiting examples of viruses that are derived from attenuated LIVPviruses, such as GLV-1h68, and that are therapeutic viruses for whichtherapy can monitored, include, but are not limited to, LIVP virusesdescribed in U.S. Pat. Nos. 7,588,767, 7,588,771, 7,662,398 and7,754,221 and U.S. Patent Publication Nos. 2007/0202572, 2007/0212727,2010/0062016, 2009/0098529, 2009/0053244, 2009/0155287, 2009/0117034,2010/0233078, 2009/0162288, 2010/0196325 and 2009/0136917, which areincorporated herein by reference in their entirety. For example, thevaccinia virus can be selected from among GLV-1h22, GLV-1h68, GLV-1i69(SEQ ID NO:84), GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74,GLV-1h81, GLV-1h82, GLV-1h83, GLV-1h84, GLV-1h85, or GLV-1h86, which aredescribed in U.S. Patent Publication No. 2009/0098529 and GLV-1h104,GLV-1h105, GLV-1h106, GLV-1h107, GLV-1h108 and GLV-1h109, which aredescribed in U.S. Patent Publication No. 2009/0053244; GLV-1h99,GLV-1h100, GLV-1h101, GLV-1h139, GLV-1h146, GLV-1h151, GLV-1h152 andGLV-1h153, which are described in U.S. Patent Publication No.2009/0117034.

Exemplary of viruses which have one or more expression cassettes removedfrom GLV-1h68 and replaced with a heterologous non-coding DNA moleculeinclude GLV-1h70, GLV-1h71, GLV-1h72, GLV-1h73, GLV-1h74, GLV-1h85, andGLV-1h86. GLV-1h70 contains (P_(SEL))Ruc-GFP inserted into the F14.5Lgene locus, (P_(SEL))rTrfR and (P_(7.5k))LacZ inserted into the TK genelocus, and a non-coding DNA molecule inserted into the HA gene locus inplace of (P_(11k))gusA. GLV-1h71 contains a non-coding DNA moleculeinserted into the F14.5L gene locus in place of (P_(SEL))Ruc-GFP,(P_(SEL))rTrfR and (P_(7.5k))LacZ inserted into the TK gene locus, and(P_(11k))gusA inserted into the HA gene locus. GLV-1h72 contains(P_(SEL))Ruc-GFP inserted into the F14.5L gene locus, a non-coding DNAmolecule inserted into the TK gene locus in place of (P_(SEL))rTrfR and(P_(7.5k))LacZ, and P_(11k)gusA inserted into the HA gene locus.GLV-1h73 contains a non-coding DNA molecule inserted into the F14.5Lgene locus in place of (P_(SEL))Ruc-GFP, (P_(SEL))rTrfR and(P_(7.5k))LacZ inserted into the TK gene locus, and a non-coding DNAmolecule inserted into the HA gene locus in place of (P_(11k))gusA.GLV-1h74 contains a non-coding DNA molecule inserted into the F14.5Lgene locus in place of (P_(SEL))Ruc-GFP, a non-coding DNA moleculeinserted into the TK gene locus in place of (P_(SEL))rTrfR and(P_(7.5k))LacZ, and a non-coding DNA molecule inserted into the HA genelocus in place of (P_(11k))gusA. GLV-1h85 contains a non-coding DNAmolecule inserted into the F14.5L gene locus in place of(P_(SEL))Ruc-GFP, a non-coding DNA molecule inserted into the TK genelocus in place of (P_(SEL))rTrfR and (P_(7.5k))LacZ, and (P_(11k))gusAinserted into the HA gene locus. GLV-1h86 contains (P_(SEL))Ruc-GFPinserted into the F14.5L gene locus, a non-coding DNA molecule insertedinto the TK gene locus in place of (P_(SEL))rTrfR and (P_(7.5k))LacZ,and a non-coding DNA molecule inserted into the HA gene locus in placeof (P_(11k))gusA

Other exemplary viruses include, but are not limited to, LIVP virusesthat express one or more therapeutic gene products, such as angiogenesisinhibitors (e.g., GLV-1h81, which contains DNA encoding the plasminogenK5 domain under the control of the vaccinia synthetic early-latepromoter in place of the gusA expression cassette at the HA locus inGLV-1h68; GLV-1h104, GLV-1h105 and GLV-1h106, which contain DNA encodinga truncated human tissue factor fused to the α_(v)β₃-integrin RGDbinding motif (tTF-RGD) under the control of a vaccinia synthetic earlypromoter, vaccinia synthetic early/late promoter or vaccinia syntheticlate promoter, respectively, in place of the LacZ/rTFr expressioncassette at the TK locus of GLV-1h68; GLV-1h107, GLV-1h108 andGLV-1h109, which contain DNA encoding an anti-VEGF single chain antibodyG6 under the control of a vaccinia synthetic early promoter, vacciniasynthetic early/late promoter or vaccinia synthetic late promoter,respectively, in place of the LacZ/rTFr expression cassette at the TKlocus of GLV-1h68) and proteins for tumor growth suppression (e.g.,GLV-1h90, GLV-1h91 and GLV-1h92, which express a fusion proteincontaining an IL-6 fused to an IL-6 receptor (sIL-6R/IL-6) under thecontrol of a vaccinia synthetic early promoter, vaccinia syntheticearly/late promoter or vaccinia synthetic late promoter, respectively,in place of the gusA expression cassette at the HA locus in GLV-1h68;and GLV-1h96, GLV-1h97 and GLV-1h98, which express IL-24 (melanomadifferentiation gene, mda-7) under the control of a vaccinia syntheticearly promoter, vaccinia synthetic early/late promoter or vacciniasynthetic late promoter, respectively, in place of the Ruc-GFP fusiongene expression cassette at the F14.5L locus of GLV-1h68). Additionaltherapeutic gene products that can be engineered in the viruses providedherein also are described elsewhere herein.

Exemplary transporter proteins that can be encoded by the virusesprovided herein include, for example, the human norepinephrinetransporter (hNET) and the human sodium iodide symporter (hNIS).Exemplary viruses that can be employed in the methods and use providedherein that encode the human norepinephrine transporter (hNET) include,but are not limited to, GLV-1h99, GLV-1h100, GLV-1h101, GLV-1h139,GLV-1h146, and GLV-1h150. GLV-1h99 encodes hNET under the control of avaccinia synthetic early promoter in place of the Ruc-GFP fusion geneexpression cassette at the F14.5L locus of GLV-1h68. GLV-1h100 andGLV-1h101 encode hNET under the control of a vaccinia synthetic earlypromoter or vaccinia synthetic late promoter, respectively, in place ofthe LacZ/rTFr expression cassette at the TK locus of GLV-1h68. GLV-1h139encodes hNET under the control of a vaccinia synthetic early promoter inplace of the gusA expression cassette at the HA locus in GLV-1h68.GLV-1h146 and GLV-1h150, encode hNET under the control of a vacciniasynthetic early promoter or vaccinia synthetic late promoter,respectively, in place of the LacZ/rTFr expression cassette at the TKlocus of GLV-1h100 and GLV-101, respectively. Thus, GLV-1h146 andGLV-1h150 encode both hNET and IL-24. Exemplary viruses that can beemployed in the methods and use provided herein that encode the humansodium iodide transporter (hNIS) include, but are not limited to,GLV-1h151, GLV-1h152 and GLV-1h153. GLV-1h151, GLV-1h152 and GLV-1h153encode hNIS under the control of a vaccinia synthetic early promoter,vaccinia synthetic early/late promoter or vaccinia synthetic latepromoter, respectively, in place of the gusA expression cassette at theHA locus in GLV-1h68.

Other exemplary viruses include, but are not limited to, LIVP virusesthat encode additional imaging agents such as ferritin and/or atransferrin receptor (e.g., GLV-1h82 and GLV-1h83 which encode E. coliferritin at the HA locus; GLV-1h82 addition encodes the humantransferrin receptor at the TK locus) or a click beetle luciferase-redfluorescent protein fusion protein (e.g., GLV-1h84, which encodes CBG99and mRFP1 at the TK locus). During translation, the two proteins arecleaved into two individual proteins at picornavirus 2A element (Osbornet al., (2005) Mol. Ther. 12: 569-574). CBG99 produces a more stableluminescent signal than does Renilla luciferase with a half-life ofgreater than 30 minutes, which makes both in vitro and in vivo assaysmore convenient. mRFP 1 provides improvements in in vivo imagingrelative to GFP since mRFP1 can penetrate tissue deeper than GFP.

ii. Other Cytoplasmic Viruses

Other therapeutic viruses whose therapy can be detected and monitored asdescribed herein include cytoplasmic viruses that are not poxviruses. Avariety of such cytoplasmic viruses are known in the art, and includeAfrican swine flu family viruses and various RNA viruses such asarenaviruses, picornaviruses, caliciviruses, togaviruses, coronaviruses,paramyxoviruses, flaviviruses, reoviruses, and rhaboviruses. Exemplarytogaviruses include Sindbis viruses. Exemplary arenaviruses includelymphocytic choriomeningitis virus. Exemplary rhaboviruses includevesicular stomatitis viruses. Exemplary paramyxoviruses includeNewcastle Disease viruses and measles viruses. Exemplary picornavirusesinclude polio viruses, bovine enteroviruses and rhinoviruses. Exemplaryflaviviruses include Yellow fever virus; attenuated Yellow fever virusesare known in the art, as exemplified in Barrett et al. (Biologicals 25:17-25 (1997)), and McAllister et al. (J. Virol. 74: 9197-9205 (2000)).

Also provided for use as a biological therapeutic herein are modifiedviruses that have one or more enhanced characteristics relative to thewild type virus. Such characteristics can include, but are not limitedto, attenuated pathogenicity, reduced toxicity, preferentialaccumulation in tumor, increased ability to activate an immune responseagainst tumor cells, increased immunogenicity, increased or decreasedreplication competence, and are able to express exogenous proteins, andcombinations thereof. In some embodiments, the modified viruses have anability to activate an immune response against tumor cells withoutaggressively killing the tumor cells. In other embodiments, the virusescan be modified to express one or more detectable genes, including genesthat can be used for imaging. In other embodiments, the viruses can bemodified to express one or more genes for harvesting the gene productsand/or for harvesting antibodies against the gene products.

iii. Adenovirus, Herpes, Retroviruses

Other viruses for use as a biological therapeutic in the methods hereininclude viruses that include in their life cycle entry of a nucleic acidmolecule into the nucleus of the host cell. A variety of such viruses isknown in the art, and includes herpesviruses, papovaviruses,retroviruses, adenoviruses, parvoviruses and orthomyxoviruses. Exemplaryherpesviruses include herpes simplex type 1 viruses, cytomegaloviruses,and Epstein-Barr viruses. Exemplary papovaviruses include humanpapillomavirus and SV40 viruses. Exemplary retroviruses includelentiviruses. These viruses have been employed in a variety of genetherapy can be cell-based therapeutic methods. Exemplaryorthomyxoviruses include influenza viruses. Exemplary parvovirusesinclude adeno associated viruses. Adenoviruses have been employed forcell therapy. In addition, adenoviruses, such as the onyx viruses andothers, have been modified, such as by deletion of EA1 genes, so thatthey replicate in cancerous cells, and, thus, are oncolytic.Adenoviruses also have been engineered to have modified tropism fortumor therapy and also as gene therapy vectors. Oncolytic viruses foruse as a biological therapeutic in the methods provided herein are wellknown to one skill in the art and include, for example, vesicularstomatitis virus, see, e.g., U.S. Pat. Nos. 7,731,974, 7,153,510,6,653,103 and U.S. Pat. Pub. Nos. 2010/0178684, 2010/0172877,2010/0113567, 2007/0098743, 20050260601, 20050220818 and EP Pat. Nos.1385466, 1606411 and 1520175; herpes simplex virus, see, e.g., U.S. Pat.Nos. 7,897,146, 7,731,952, 7,550,296, 7,537,924, 6,723,316, 6,428,968and U.S. Pat. Pub. Nos. 2011/0177032, 2011/0158948, 2010/0092515,2009/0274728, 2009/0285860, 2009/0215147, 2009/0010889, 2007/0110720,2006/0039894 and 20040009604; retroviruses, see, e.g., U.S. Pat. Nos.6,689,871, 6,635,472, 5,851,529, 5,716,826, 5,716,613 and U.S. Pat. Pub.No. 20110212530; and adeno-associated viruses, see, e.g., U.S. Pat. Nos.8,007,780, 7,968,340, 7,943,374, 7,906,111, 7,927,585, 7,811,814,7,662,627, 7,241,447, 7,238,526, 7,172,893, 7,033,826, 7,001,765,6,897,045, and 6,632,670.

b. Bacteria

The biological therapy monitored in the methods herein can be bacteria,such as an attenuated or non-pathogenic bacterium, that accumulates atsites of cellular proliferation, including tumors, tumor tissues,metastases, areas of inflammation, immunoprivileged sites or tissues,wounds and/or infections (see, e.g., U.S. Pat. Nos. 7,763,420,7,820,184, 7,514,089, 7,452,531, 7,354,592, 6,962,696, 6,923,972,6,863,894, 6,685,935, 6,475,482, 7,687,474, 7,255,851 and 6,638,752;U.S. Pat. Publ. Nos. 2003/0059400, 2004/0234455, 2005/0069491,2009/0117049, 2009/0117048, 2009/0117047, 2009/0123382, 2003/0228261,2004/0213741, 2005/0249670, 2011/0223241, 2010/0136048, 2009/0169517,2008/0124355 and 2007/0009489; and Pawelek et al., (2003) Lancet Oncol4:528-556, King et al., (2002) Hum Gene Ther 13:1225-1233, Soghomonyanet al., (2005) Cancer Gene Ther 12:101-108, Friedlos et al., (2008) ClinCancer Res 14:4259-4266, King et al., (2009) Methods Mol Biol542:649-659, Kapoor et al., (2011) Antimicrob Agents Chemother55:3058-3062, Barak et al., (2010) BMC Cancer 10:146, and Contag (2007)Annnu Rev Pathol 2:277-305. Thus, the bacteria are used to treatdiseases and disorders, including, for example, proliferativeconditions, neoplastic diseases, tumors, tumor tissue, cancer,metastasis, inflammation, wounds and infections. The bacterium used inthe methods provided herein can be modified to express the reporterprotein, e.g., reporter enzyme, and additionally can be modified tocontain one or more additional exogenous genes. In some examples, thebacterium is modified to contain one or more regulatory sequences toregulate expression of an exogenous gene.

Generally, the therapeutic bacteria monitored in the methods herein areattenuated or non-pathogenic, and they generally replicate in the hostor target tissue. Bacteria include mutual, commensal and/or probioticstrains of bacteria. For example the bacteria include strains ofbacteria that coexist in a commensal or mutualistic relationship with asubject such as, for example, an animal, including human and non-humananimals. Exemplary bacteria for use in the methods include mutual,commensal and/or probiotic strains of Escherichia coli, Bacteroides,Eubacterium, Streptococcus, Actinomyces, Veillonella, Nesseria,Prevotella, Campylobacter, Fusobacterium, Eikenella, Porphyromonas andPriopionibacteria. Exemplary of probiotic bacteria are Escherichia colistrain Nissle 1917. Other exemplary probiotic strains include, but arenot limited to, Bacillus cereus, Bacillus licheniformis, Bacilluspumilus, Bacillus clausii, Bacillus coagulans, Bacillus polyfermenticus,Brevibacillus laterosporus, Lactococcus, Lactobacillus reuteri,Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillusgallinarum, Lactobacillus gasseri, Lactobacillus johnsonii,Lactobacillus bifidum, Lactobacillus helveticus, Bifidobacterium lactis,Bifidobacterium breve, Leuconostoc mesenteroides, Enterococcus faecium,Pediococcus and Sporolactobacillus inulinu. Other exemplary bacteria foruse in the methods provided herein include strains that are notprobiotic, including but not limited to, Clostridia, Salmonella,Shigella, Bifidobacteria and Staphylococci.

Therapeutic bacteria are those that tend to accumulate in a certain areaor areas of a subject to whom the bacteria are administered. Bacteriaused in the methods typically are capable of selectively accumulating inproliferative sites or condition (including, for example, a tumor, tumortissue, cancer, metastasis, neoplasm, neoplastic disease, site ofinflammation, wound, wound tissue and infection) or in immunoprivilegedsites relative to other locations in a subject. Because bacteriaselectively accumulate at such sites, they can be used to specificallydeliver substances and compositions to the sites, including therapeuticsubstances and compositions for use in treating diseases, disorders andconditions associated with proliferation sites and conditions,including, for example, tumors, cancers, neoplasms, neoplastic diseases,inflammation, wounds and other diseases, conditions and disorders asdescribed herein. Also there are bacteria that provide a therapeuticbenefit in the treatment of diseases, disorders and/or conditionswithout necessarily providing for delivery of a separate therapeuticsubstance or composition. These include, but are not limited, toprobiotic bacteria such as the Nissle strain of E. coli.

The probiotic bacteria monitored by the methods herein can be modifiedfrom their wild-type form. Modifications can include any of a variety ofchanges, and typically include changes to the genome or nucleic acidmolecules of the bacteria. For example, modifications of bacteria caninclude one or more modifications of the bacterial genome to add, deleteor replace nucleic acid. Such modifications can alter one or moreproperties of the bacteria including, but not limited to, pathogenicity,toxicity, ability to preferentially accumulate in tumors, ability tolyse cells or cause cell death, replication competence, increasedcapacity to capture iron or other metals, increased capacity totransport iron, increased capacity to store iron, bind a ligand, or acombination thereof. Exemplary modifications include, but are notlimited to deletion of one or more endogenous genes, addition of one ormore exogenous genes, mutation of one or more endogenous gene productsor alteration of gene expression of one or more endogenous genes.

In some examples, the bacterium can be modified to express one or moreexogenous genes in addition to the reporter protein. Exemplary exogenousgene products include proteins and RNA molecules. The modified bacteriacan express gene products that are useful for diagnostic or therapeuticuses. Exemplary exogenous gene products that can be expressed by themodified bacteria include, but are not limited to, a detectable geneproduct (e.g., fluorescent proteins, luminescent proteins), a geneproduct that induces a detectable signal (e.g. luciferases, ferritin,siderophore), a therapeutic gene product, a protein that serves as abinding site for a ligand (e.g., receptors (e.g., transferrin receptor)or other transmembrane or membrane associated proteins), proteins usefulfor tumor therapy (e.g., Pseudomonas A endotoxin, diphtheria toxin, p53,Arf, Bax, tumor necrosis factor alfa, HSV TK, E. coli purine nucleosidephosphorylase and derivatives thereof, cytosine deaminases, uracil,phosphoribosyltranspherase and fusions thereof (e.g. FCU1), angiostatin,endostatin, different cytokines) and many other proteins.

In some examples, the bacterium is modified to contain one or moreregulatory sequences to regulate expression of the exogenous gene. As isknown in the art, regulatory sequences can permit constitutiveexpression of the exogenous gene or can permit inducible expression ofthe exogenous gene. Further, the regulatory sequence can permit controlof the level of expression of the exogenous gene. In some examples,inducible expression can be under the control of cellular or otherfactors present in a tumor cell, present in a bacterially-infected tumorcell, or present in/on extracellular bacteria localized in a tumorenvironment. In other examples, inducible expression can be under thecontrol of an administrable substance, including sugars such asarabinose, xylose, IPTG, RU486 or other known induction compounds. Anyof a variety of regulatory sequences are available to one skilled in theart according to known factors and design preferences. In some examples,the regulatory sequence can result in constitutive, high levels of geneexpression. In tumor therapy examples, a therapeutic protein can beunder the control of an internally inducible promoter or an externallyinducible promoter. In some examples, the inducible promoter is asugar-inducible promoter, such as an arabinose- or xylose-induciblepromoter. Recombinant bacteria that contain a sugar-inducible promoterfor the expression of exogenous genes can be modified to decrease orabolish the metabolic breakdown of the inducing sugar. For example,bacteria, such as E. coli, can be modified such that the breakdownand/or utilization of arabinose in the bacteria is reduced or abolished,which allows for greater accumulation of arabinose in the cells leadingto higher gene induction of and longer gene expression fromarabinose-inducible promoters in the recombinant bacteria. In oneexample of the methods provided, inducible promoters can be used toinitiate expression of a gene product once the bacteria have accumulatedto a particular concentration at the accumulation.

i. E. coli Strain Nissle 1917

Exemplary of a bacteria that can be used as a biological therapeutic inthe provided methods is E. coli strain Nissle 1917, as described inSchultz et al. (2005) J. Microbiol. Methods 61(3): 389-398 and U.S. Pat.Nos. 7,763,420 and 7,820,184 and U.S. Patent Publication No.2009/0180955, all of which are incorporated herein by reference. E. colistrain Nissle 1917 lacks defined virulence factors such asalpha-hemolysin and other toxins, mannose-resistant hemagglutinatingadhesins, P-fimbrial adhesins, and the semi-rough lipopolysaccharidephenotype (Blum et al. (1996) Infection. 23(4):234-236). The unique LPSstructure furthermore contributes to its decreased immunotoxicity whilemaintaining serum sensitivity. Serum sensitivity can contribute toselective colonization of Nissle 1917 in immunoprivileged areas such astumors, since the bacteria colonize those sites, such as tumors, thatare sequestered from the immune system. Nissle 1917 possesses enhancedproperties for its use as a therapeutic in part due to the expression ofat least six different iron uptake systems, including siderophores suchas aerobactin, salmochelin, enterobactin, and yersiniabactin; chu hemetransport locus and a ferric dicitrate transport system. The lack ofpathogenicity and probiotic properties have lead to its use for thetreatment of gut disorders, such as ulcerative colitis, chronicconstipation, Crohn's disease, pouchitis, irritable bowel syndrome, andother forms of colitis and gut perturbations. The fact that itaccumulates in tumor cells and also can participate in uptake ofdetectable compounds for imaging and/or therapeutic compounds fortreatment has led to its use treating tumors.

E. coli strain Nissle 1917 bacteria used in the methods provided hereincan be modified. Exemplary nucleic acid molecular modifications includetruncations, insertions, deletions and mutations. In an exemplarymodification, a microorganism or cell can be modified by truncation,insertion, deletion or mutation of one or more genes. In an exemplaryinsertion, an exogenous gene can be inserted into the genome of themicroorganism or cell or provided on a plasmid. In an exemplarymodification, an endogenous gene, an exogenous gene or a combinationthereof can be inserted into a plasmid which is inserted into the E.coli strain Nissle 1917 bacterium using any of the methods known in theart.

ii. Other Bacteria

Exemplary bacteria that can be used in the methods provided hereininclude, but are not limited to, strains Clostridia, Salmonella,Shigella, Bifidobacteria and Staphylococci. In one example, the bacteriafor use in the methods provided herein is a Salmonella typhimurium, suchas S. typhimurium strain SL7838, S. typhimurium strain VNP20009 and S.typhimurium strain TAPET-CD (see, e.g, Barak et al., (2010) BMC Cancer10:146, Friedlos et al., (2008) Clin Cancer Res 14:4259-4266, Low etal., (2004) Methods Mol Med 90:47-60, Soghomonyan et al., (2005) CancerGene Ther 12:101-108 and King et al., (2009) Methods Mol Biol542:649-659). Bifidobactera for use in the methods provided hereininclude, but are not limited to, B. infantis (Zhu et al., (2011) CancerGene Ther 18:884-896), B. breve (Cronin et al., (2010) Mol Ther18:1397-1407), B. adolescentis (Hu et la., (2009) Cancer Gene Ther16:655-663) and B. longum (Taniguchi et al., (2010) Cancer Sci101:1952-1932). Clostridia for use in the methods provided hereininclude, but are not limited to, C. perfringens (Li et al., (2009) HumanGene Ther 20:751-758, Li et al., (2008) J Natl Cancer Inst100:1389-1400) and C. butyricum (Mose et la., (1964) Cancer Res24:212-216), C. acetobutylicum (Barbe et al., (2005) FEMS Microbiol Lett246:67-73, Theys et al., (2001) Cancer Detect Prev 25:548-557) and C.novyi-NT (Wei et al., (2008) Cancer Lett 259:16-27).

c. Other Biological Therapies

As noted, the methods herein can be used to monitor therapy with anybiological therapeutic in which a gene product is produced. In someexamples, the biological therapies include, but are not limited to, genetherapies, immunotherapies, adoptive immunotherapies and other celltherapies.

i. Gene Therapy

In some examples, the method provided herein is used to detectcolonization or replication of a gene therapy vector. Gene therapy orgenetic therapy involves the transfer of nucleic acid, such as DNA, intocertain cells, e.g., target cells, of a mammal, particularly a human,with a disorder or condition for which such therapy is sought. Thenucleic acid, such as DNA, is introduced into the selected target cells,such as directly or in a vector or other delivery vehicle, in a mannersuch that the heterologous nucleic acid, such as DNA, is expressed and atherapeutic product encoded thereby is produced resulting inamelioration or elimination the symptoms, or manifestations of aninherited or acquired disease or curing of the disease. Alternatively,the heterologous nucleic acid, such as DNA, can in some manner mediateexpression of DNA that encodes the therapeutic product, or it can encodea product, such as a peptide or RNA that in some manner mediates,directly or indirectly, expression of a therapeutic product. Genetictherapy also can be used to deliver nucleic acid encoding a gene productthat replaces a defective gene or supplements a gene product produced bythe mammal or the cell in which it is introduced. The introduced nucleicacid can encode a therapeutic compound, such as a growth factor orinhibitor thereof, or a tumor necrosis factor or inhibitor thereof, suchas a receptor therefore, that is not normally produced in the mammalianhost or that is not produced in therapeutically effective amounts or ata therapeutically useful time. The heterologous nucleic acid, such asDNA, encoding the therapeutic product can be modified prior tointroduction into the cells of the afflicted host in order to enhance orotherwise alter the product or expression thereof. Genetic therapy alsocan involve delivery of an inhibitor or repressor or other modulator ofgene expression.

Typically, for detection by the methods provided herein, gene therapyinvolves administration of a vector encoding the reporter protein to asubject. The vector can any vector, including prokaryotic and mammalianvectors, such as viral vectors, mammalian vectors, bacterial vectors,insect vectors, plant vectors and artificial chromosomes. In someexamples, the vector is administered in a virus, such as, but notlimited to, a retrovirus, adenovirus, adeno-associated virus and herpessimplex virus. In other examples, the vector is administered in aliposome, PEGylated liposome, nanoparticle, lipid-based nanoparticle orlymphocyte. In yet other example, the vector is delivered administereddirectly to the subject. In other examples of gene therapy used in themethods provided herein, biologically active nucleic acid molecules,such as RNAi and antisense, are administered to a subject.

ii. Cell Therapy

In some examples, the method provided herein is used to detectcolonization or replication of cells introduced for cell therapy. Celltherapy includes cell transplants, including, but not limited to,pancreatic islet, bone marrow, endothelial, epidermal, myoblast, neuraland stem cell transplants. Typically, the cell therapy contains a vectorencoding a reporter protein. In some examples, the vector contained inthe cell therapy encodes an additional heterologous protein, such as atherapeutic protein. A vector for use in a cell therapy provided hereincan be a viral vector, mammalian vector, bacterial vector, insectvector, plant vector or artificial chromosome encoding the reportergene. In some examples, the cell is infected with a virus encoding areporter protein prior to administration to the subject.

iii. Immunotherapy

In some examples, the methods provided herein are used to detectcolonization or replication of colonization/replication of cells used inimmunotherapy or adoptive immunotherapy. Immunotherapy is used tostimulate the immune system, for example, to stimulate the immune systemto eliminate cancer cells. Active immunotherapy involves injection ofcells or proteins, for example, cancer or tumor cells, to generateeither new or enhance systemic immune responses to the administered cellor protein. Passive immunotherapy involves the administration of anantibody. Adoptive immunotherapy is a therapeutic approach for treatingcancer or infectious diseases in which immune cells are administered toa host with the aim that the cells mediate either directly or indirectlyspecific immunity to tumor cells and/or antigenic components orregression of the tumor or treatment of infectious diseases, as the casecan be. As used in the methods provided herein, immunotherapy oradoptive immunotherapy can include administration of antibodies,proteins or cells for either active or passive immunotherapy. In someexamples, the proteins are contained in nanoparticles. In otherexamples, the immunotherapy involves administration of immune cells or avaccine. The methods herein, detect, in distinct loci from the targetedtissues or cells, expression of a protein introduced by the therapy orencoded thereby.

2. Reporter Proteins

The biological therapeutics for use in the methods provided herein canencode a reporter protein, which is a detectable protein or a proteinthat induces a detectable signal. The reporter protein can be endogenousto the therapeutic, such as an endogenous detectable or enzymatic viralproduct, or can be heterologous. The reporter protein can be a productthat normally occurs in the host, in which case increased expression isassessed, or it can be a non-native product, in which any amount ofexpression is detected, or it can be a product not normally expressed inthe sample that is tested.

Upon expression of the reporter gene, the protein or a signal induced bythe protein can be detected. Typically, the reporter genes for use inthe methods provided herein encode enzymes that catalyze the reaction ofa substrate into a detectable product. For example, enzyme reportergenes can catalyze the reaction of a substrate to produce a detectableproduct or signal, such as a fluorescent, luminescent, chromogenic orspectrophotometric response. Any enzyme is contemplated as long as theprotein is capable of being expressed in the biological therapeutic, forexample, the oncolytic virus or bacteria. In one example, enzymaticreporter proteins used in the methods herein are human enzymes,including mitochondrial enzymes. In another example, enzymatic reporterproteins used in the methods provided herein are prokaryotic, insect, orplant enzymes. Exemplary reporter enzymes that can be used in theprovided method include, but are not limited to, β-glucuronidases,luciferases, beta-galactosidases, chloramphenicol acetyltransferases(CAT) and alkaline phosphatases.

A reporter protein substrate is any substrate upon which interactionwith the reporter protein induces a product or signal that can bedetected. The signal can be detected directly or indirectly. Typically,the reporter proteins are enzymatic reporter proteins that catalyzeproduction of a product from a substrate. A reporter enzyme substrate isany substrate, for example, any compound, that is a substrate for thereporter enzyme. A reporter enzyme is capable of reacting with thesubstrate causing a change in the substrate, or a signal, that can bemonitored or detected either directly or indirectly. Typically, areporter enzyme substrate is a compound that is modified upon reactionwith the reporter enzyme resulting in a modified compound that can bedirectly monitored. For example, reporter enzyme substrates typicallyare colorless or non-fluorescent substrates or compounds that aretransformed into colored or fluorescent products upon reaction with thereporter enzyme. For example, in the method provided herein, a reporterenzyme substrate can be a fluorescent, luminescent, chromogenic orspectrophotometric substrate. Reporter enzyme substrates are well knownand it is understood that a person of skill in the art can select asuitable substrate for use in the methods provided herein.

a. Reporter Enzymes

Enzymatic reporter genes that can be used in the provided methodsinclude, but are not limited, any protein that exhibits enzymaticactivity, e.g., lipases, phospholipases, sulfatases, ureases,peptidases, proteases, esterases, phosphatases, acid phosphatases,glycosidases, glucosidases, glucuronidases, galactosidases,carboxylesterases, luciferases, peroxidases, hydrolases,oxidoreductases, lyases, transferases, isomerases, ligases, synthases,protein kinases, esterases, isomerases, glycosylases, synthetases,dehydrogenases, oxidases, reductases, methylases, oxidases, P450enzymes, monoamine oxidases (MAOs), flavin monoamine oxidases (FMOs),transferases, glutathione S transferases (GSTs), xanthineguaninephosphoribosyl-transferase, alkaline phosphatases (AP), invertases,luciferases, acetyltransferases, beta-glucuronidases, exo-glucanases,glucoamylases, beta-glucosidases, horseradish peroxidases, alkalinephosphatases, beta-lactamases, alpha-amylases, alpha-glucosidases,catalases, beta-xylosidases, beta-galactosidases, chondroitinsulfatases,gelatinases, collagenases, caseinases, nitroreductases, azoreductases,demethylases, deacetylases, deformylases, phosphatases, kinases,peroxidases, sulfotases, acetylcholinesterases, dehydrogenases,dealkylases and oxygenases. The enzyme reporter genes employed in themethods encode both recombinant and endogenous (native) enzymes. In oneembodiment, the enzyme reporter gene encodes an endogenous enzyme. Inanother embodiment, the enzyme reporter gene encodes a recombinantenzyme. Enzyme reporter genes can easily be determined by one ofordinary skill in the art.

In one embodiment, the enzyme reporter gene encodes a hydrolytic enzyme.Examples of hydrolytic enzymes include alkaline and acid phosphatases,esterases, decarboxylases, phospholipase D, P-xylosidase,β-D-fucosidase, thioglucosidase, β-D-galactosidase, α-D-galactosidase,α-D-glucosidase, β-D-glucosidase, glucuronidase, β-D-mannosidase,β-D-mannosidase, β-D-fructofuranosidase, and β-D-glucosiduronase.Exemplary enzymatic reporter proteins that can be used in the methodsprovided herein, include, but are not limited to, β-glucuronidases,β-galactosidases, luciferases, chloramphenicol acetyltransferases (CAT)and alkaline phosphatases.

i. β-Glucuronidases

Beta-glucuronidases (β-glucuronidases; Gus; EC 3.2.1.31) are members ofthe glycosidase family of enzymes that catalyze the breakdown of complexcarbohydrates. Beta-glucuronidases are part of the glycosyl hydrolase 2family of glycosidases. Beta-glucuronidases catalyze the hydrolysis ofβ-D-glucuronides into the corresponding D-glucuronate and alcohol.

Human β-glucuronidase catalyzes the hydrolysis of β-D-glucuronic acidresidues from the non-reducing end of mucopolysaccharides, such asheparin sulfate. Human β-glucuronidase is found in the lysosome where itconverts conjugated bilirubin into an unconjugated form forreabsorption. Human β-glucuronidase is also found in breast milk, andcontributes to neonatal jaundice. The gene encoding β-glucuronidase islocated on chromosome 7. The β-glucuronidase transcript (SEQ ID NO:1) isnormally translated to form a 651 amino acid precursor polypeptide (SEQID NO:5) containing a 22 amino acid signal sequence at the N-terminus(amino acid residues 1-22). The mature β-glucuronidase therefore, is a629 amino acid polypeptide set forth in SEQ ID NO:121 (Oshima et al.,(1987) Proc Natl Acad Sci USA 84:685-689). Beta-glucuronidase isN-linked glycosylated with 3-4 oligosaccharide chains at residues N173,N272, N420 and N631 of SEQ ID NO:5.

Structural analysis reveals human β-glucuronidase is synthesized as an80 kDa monomer that undergoes proteolysis in the lysosome to remove asignal sequence from the C-terminal end to form a 78 kDa monomer (Islamet al., (1993) 268:22627-22633; Shipley et al., (1993) J Biol Chem268:12193-12198). Biologically, β-glucuronidase exists as a 332 kDahomotetramer (Kim et al., (2008) Acta Crystallogr Sect F Struct BiolCryst Commun 64(Pt. 12):1169-1171). The tetramer has approximatedihedral symmetry and each promoter includes three structural domainswith topologies similar to a jelly roll barrel, an immunoglobulinconstant domain and a TIM barrel respectively. The active site of theenzyme is formed from a large cleft at the interface of two monomers.Residues Glu 451 and Glu 540 of SEQ ID NO:5 are proposed to be importantfor catalysis (Jain et al., (1996) Nat Struct Biol 3(4):375-381).β-cholinesterases of this type include, but are not limited to,β-cholinesterases from mouse (SEQ ID NO:115, DNA set forth in SEQ IDNO:2), rat (SEQ ID NO:114, DNA set forth in SEQ ID NO:6), dog (SEQ IDNO:116, DNA set forth in SEQ ID NO:7), cat (SEQ ID NO:117, DNA set forthin SEQ ID NO:8), pig (SEQ ID NO:120, DNA set forth in SEQ ID NO:11),green monkey (SEQ ID NO:118, DNA set forth in SEQ ID NO:9) and Sumatranorangutan (SEQ ID NO:119, DNA set forth in SEQ ID NO:10). Mammalianβ-glucuronidases with a pH-optimum under acidic conditions (pH 4 to 5)have strongly reduced capacity at normal (neutral) tissue pH, whereas E.coli β-glucuronidase encoded by gusA works optimal in the range of pH6.8 to 7.7 (Fang et al., (1995) Vet Microbiol 46:361-367).

β-glucuronidases occur in various bacteria, including, but not limitedto, E. coli, Shigella, Salmonella, Lactobacillus, Streptococcus,Clostridium, Roseburia, Anaerococcus, Victivallis, Congregibacter andAspergillus (Kilian and Bulow (1976) Acta Pathol Microbiol Scand B.84B(5):245-251). β-glucuronidase from E. coli strain K12 (gene set forthin SEQ ID NO:3) is normally translated into a 603 amino acid protein setforth in SEQ ID NO:4. Residue E413 of SEQ ID NO:4 is an active siteproton donor. Bacterial β-glucuronidases of this type include, but arenot limited to, β-glucuronidases from E. coli K12 (SEQ ID NO:4, DNA setforth in SEQ ID NO:3), Shigella flexneri strain K-18 (SEQ ID NO:128, DNAset forth in SEQ ID NO:127), Salmonella enterica (SEQ ID NO:136, DNA setforth in SEQ ID NO:135), Lactobacillus brevis strain RO1 (SEQ ID NO:130,DNA set forth in SEQ ID NO:129), Streptococcus agalactiae (SEQ IDNO:132, DNA set forth in SEQ ID NO:131), Clostridium perfringens (SEQ IDNO:134, DNA set forth in SEQ ID NO:133), Roseburia intestinalis (SEQ IDNO:138, DNA set forth in SEQ ID NO:137), Anaerococcus tetradius (SEQ IDNO:140, DNA set forth in SEQ ID NO:139), Victivallis vadensis (SEQ IDNO:142, DNA set forth in SEQ ID NO:141), Congregibacter litoralis (SEQID NO:144, DNA set forth in SEQ ID NO:143) and Aspergillus terreus (SEQID NO:146, DNA set forth in SEQ ID NO:145). Exemplary of aβ-glucuronidases used in the provided methods are human and bacterialβ-glucuronidases. A β-glucuronidase set forth in any of SEQ ID NOS:1-11, 114-121 and 127-146, active fragments thereof, or variants thereofcan be used in the provided methods.

Also included in the methods provided herein are variants of any of SEQID NOS:1-11, 114-121 and 127-146 that have at least or about at least orabout or 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more sequence identity to any of SEQ ID NOS: 1-11, 114-121 and127-146. Amino acid variants include variants that contain conservativeand non-conservative mutations. It is understood that residues that areimportant or otherwise required for the activity of a β-glucuronidase,such as any described above or known to skill in the art, are generallyinvariant and cannot be changed. These include, for example, active siteresidues. Thus, for example, amino acid residues 451 and 540(corresponding to residues in the human β-glucuronidase set forth in SEQID NO:5) of a human β-glucuronidase are generally invariant and are notaltered, or amino acid residue E413 (corresponding to residues in the E.coli K12 β-glucuronidase set forth in SEQ ID NO:4) of a bacterialβ-glucuronidase are not altered.

For example, there are several alternative splicing forms of humanβ-glucuronidase, including for example, a short form that does notcontain amino acid residues 305-355 of SEQ ID NO:5. β-glucuronidases foruse as reporter proteins in the methods provided herein also includevariants that have an amino acid modification and that exhibit analtered, such as improved, activity compared to an β-glucuronidase notincluding the modification. Such variants include those that contain anat least one amino acid modification that enhances the catalyticactivity of the β-glucuronidase. Such modified β-glucuronidases includeany β-glucuronidase described in the art, including, but not limited to,β-glucuronidases containing at least one mutation corresponding to P30S,C38G, G136R, P148S, E150K, D152G, D152N, L176F, R216W, L243P, Y320C,Y320S, N339S, K350N, H351Y, A354V, D362N, P364L, L376F, R382C, R382H,P408S, P415L, R435P, R477W, Y495C, Y508C, E540K, G572D, K606N, G607A,A619V, Y626H, W627C and L649P in the sequence of amino acids set forthin SEQ ID NO:5.

β-glucuronidases that can be used as a reporter protein in the methodsprovided herein include modified human beta-glucuronidases with enhancedactivity at neutral pH (Chen et al., (2008) Chem Biol 15:1277-1286).Also included are β-glucuronidases that have been fused to single chainhumanized antibodies for enhanced antibody-directed enzyme prodrugtherapy (Chen et al., (2011) Bioconjug Chem 22:938-948). β-glucuronidasesubstrates are set forth in section b below and Table 3.

ii. β-Galactosidases

Beta-galactosidases (β-galactosidases; EC 3.2.1.23) are hydrolaseenzymes that catalyze the hydrolysis of β-galactosides intomonosaccharides. β-galactosidases belong to the glycosyl hydrolase 35family. Beta-galactosidases cleave beta-linked terminal galactosylresidues from gangliosides, glycosaminoglycans, lactosylceramides,lactose, and other various glycoproteins. Beta-galactosidases alsocatalyze hydrolysis of terminal non-reducing beta-D-galactose residuesin beta-D-galactosides. The active site of β-galactosidase catalyzes thehydrolysis of its disaccharide substrate via “shallow” and “deep”binding. Monovalent potassium ions (K⁺) as well as divalent magnesiumions (Mg²) are required for the enzyme's optimal activity. Thebeta-linkage of the substrate is cleaved by a terminal carboxyl group onthe side chain of a glutamic acid.

The human β-galactosidase transcript (SEQ ID NO:12) is normallytranslated to form a 677 amino acid precursor polypeptide containing a23 amino acid signal peptide (amino acids 1-23 of SEQ ID NO:13) and a 4amino acid propeptide (amino acids 24-28 of SEQ ID NO:13). The matureform therefore is a 649 amino acid polypeptide set forth in SEQ IDNO:122 (Oshima et al., (1988) Biochem Biophys Res Commun 157:238-244,Yamamoto et al., (1990) DNA Cell Biol 9:119-127). Amino acid residueE188 is an active site proton donor and residue E268 is the nucleophileof the hydrolysis reaction (of SEQ ID NO: 13). There are 7 potentialN-linked glycosylation sites, including at residues N26, N247, N464,N498, N542, N545 and N555 of SEQ ID NO:13. N-linked glycosylation hasbeen shown at N464 and N555. B-galactosidases of this type include, butare not limited to, mouse (SEQ ID NO:29, DNA set forth in SEQ ID NO:28)and dog (SEQ ID NO:33, DNA set forth in SEQ ID NO:32).

B-galactosidases occur in various bacteria, including, but not limitedto, E. coli, Lactobacillus acidophilus, Sulfolobus solfataricus,Lactococcus lactis, Geobacillus kaustiophilus, Thermus thermophilus,Bacillus subtilis and Clostridium perfringens. Bacterial β-galactosidasefrom E. coli K12 is a 464 kDa homotetramer with 2,2,2-point symmetrythat requires magnesium or manganese and sodium cofactors for activity.Each unit of β-galactosidase contains five domains, the third of whichis an active site domain (Matthews (2005) C R Biol. 328:549-556). Theenzyme can be split in two peptides, LacZα and LacZΩ, neither of whichis active by itself but both spontaneously reassemble into a functionalenzyme. E. coli K12 β-galactosidase (SEQ ID NO:15) is normallytranslated to form a 1024 amino acid polypeptide (SEQ ID NO:14) fromwhich Met1 is removed to form a 1023 amino acid mature protein. Aminoacids 538-541 of SEQ ID NO:14 form the substrate binding site, with anactive site proton donor at residue 462, a nucleophile at residue 538,sodium binding sites at residues 202, 602 and 605, magnesium 1 bindingsite at residues 417, 419 and 462 and a magnesium 2 binding site atresidue 598. Bacterial β-galactosidases of this type include, but arenot limited to, β-galactosidases from E. coli K12 (SEQ ID NO:14, DNA setforth in SEQ ID NO:15), Lactobacillus acidophilus (SEQ ID NO:16, DNA setforth in SEQ ID NO:17), Sulfolobus solfataricus (SEQ ID NO:18, DNA setforth in SEQ ID NO:19), Lactococcus lactis (SEQ ID NO:20, DNA set forthin SEQ ID NO:21), Geobacillus kaustiophilus (SEQ ID NO:22, DNA set forthin SEQ ID NO:23), Thermus thermophilus (SEQ ID NO:24, DNA set forth inSEQ ID NO:25), Bacillus subtilis (SEQ ID NO:26, DNA set forth in SEQ IDNO:27) and Clostridium perfringens (SEQ ID NO:30, DNA set forth in SEQID NO:31). Exemplary of a β-galactosidase is E. coli β-galactosidase(set forth in SEQ ID NO:13). A β-galactosidase set forth in any of SEQID NOS:12-33 and 122, active fragments thereof, or variants thereof canbe used in the methods provided herein.

Also included in the methods provided herein are variants of any of SEQID NOS:12-33 and 122 that have at least or about at least or about or60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 12-33 and 122. Amino acidvariants include variants that contain conservative and non-conservativemutations. It is understood that residues that are important orotherwise required for the activity of a β-galactosidase, such as anydescribed above or known to skill in the art, are generally invariantand cannot be changed. These include, for example, active site residues.Thus, for example, amino acid residues 188 and 268 (corresponding toresidues in the human β-galactosidase set forth in SEQ ID NO:13) of ahuman β-galactosidase are generally invariant and are not altered, oramino acid residues 462 and 538 (corresponding to residues in the E.coli K12 β-galactosidase set forth in SEQ ID NO:14) of a bacterialβ-galactosidase are not altered.

B-galactosidases also can include variants that have an amino acidmodification and that exhibits an altered, such as improved, activitycompared to a β-galactosidase not including the modification. Suchvariants include those that contain an at least one amino acidmodification that enhances the catalytic activity of theβ-galactosidase. Such modified β-galactosidases include anyβ-galactosidase described in the art, including, but not limited to,β-galactosidases containing at least one mutation corresponding to P10L,R49C, R49H, I51T, R59Q, K73E, T82M, Y83C, Y83H, H89Y, R109W, R121S,G123R, M132T, G134V, P136S, R148C, R148S, S149F, D151V, D151Y, L155R,L162S, L173P, G190D, D198Y, Y199C, R201C, R201H, R201A, D214Y, V216A,V240M, Q255H, P263S, L264S, N266S, Y270D, G272D, W273L, H281Y, Y316C,N318H, T329I, D332E, D332N, Y333H, K346N, Y347C, P297A, Q408P, T420K,T420P, L422R, S434L, L436F, G438E, D441N, R4421, Y444C, R457Q, R482C,R482H, N484K, D491N, D491Y, G494C, G494S, T5001, R521C, S532G, P549L,G554E, K578R, G579D, R590C, R590H, R595W, P597S and E632G in a humanbeta-galactosidase set forth in SEQ ID NO:13; and D202E, D202N, H358D,H358F, H358L, H358N, E462H, E538Q, H541E, H541F, H541N, F602A, G795A,E798A, E798L, E798D, E798Q, W1000F, W1000G, W1000L and W1000T in abacterial beta-galactosidase set forth in SEQ ID NO:14. B-galactosidasesubstrates are set forth in section b below and Table 3.

iii. Luciferases

Luciferases are a class of oxidative enzymes (EC 1.13.12.7) used inbioluminescence. Luciferases produce light by catalyzing the reaction ofluciferin and oxygen resulting in the production of oxyluciferin, lightand carbon dioxide. In some instances, a cofactor such as calcium ionsor ATP is necessary for the luciferase reaction. A variety of organismshave been identified that regulate light production using luciferases.For example, bacterial luciferase lux genes have been identified inPhotorhabdus luminescens and Vibrio harveyi, and eukaryotic luciferaseluc and ruc genes have been identified in firefly species (Photinussp.), green click beetle (Pyrophorus plagiophthalamus), Gaussia,railroad worm (Phrixothrix hirtus) and sea pansy (Renilla reniformis).

Luciferases that can be used in the method provided herein include, butare not limited to, luciferases from North American firefly (Photinuspyralis) (SEQ ID NO:123, DNA set forth in SEQ ID NO:124), North Americanfirefly variant I423L (Photinus pyralis) (SEQ ID NO:34, DNA set forth inSEQ ID NO:35), Southern Russian firefly (Luciola mingrelica) (SEQ IDNO:37, DNA set forth in SEQ ID NO:36), Japanese firefly (Luciolacruciata) (SEQ ID NO:39, DNA set forth in SEQ ID NO:38), Pennsylvaniafirefly (Photuris pennsylvanica) (SEQ ID NO:41, DNA set forth in SEQ IDNO:40), Sea firefly (Vargula hilgendorfii) (SEQ ID NO:43, DNA set forthin SEQ ID NO:42), Sea Pansy (Renilla reniformis) (SEQ ID NO:45, DNA setforth in SEQ ID NO:44), Green Click Beetle (Pyrophorus plagiophthalamus)(SEQ ID NO:47, DNA set forth in SEQ ID NO:46), marine copepod (Gaussiaprinceps) (SEQ ID NO:49, DNA set forth in SEQ ID NO:48) and Railroadworm (Phrixothrix hirtus) (SEQ ID NO:51, DNA set forth in SEQ ID NO:50).Bacterial Lux genes that can be included in the methods provided hereininclude Vibrio fischeri ES 114 Lux R (SEQ ID NO:93), LuxA (SEQ IDNO:94), LuxB (SEQ ID NO:95), LuxC (SEQ ID NO:96), LuxD (SEQ ID NO:97),LuxE (SEQ ID NO:98, DNA set forth in SEQ ID NO:102), LuxAB (SEQ IDNO:100), LuxCD (SEQ ID NO:101), LuxABCDE (SEQ ID NO:103) Exemplary ofluciferases that can be used in the provided methods are firefly,Renilla, Gaussia and green click beetle luciferases. Any luciferase setforth in any of SEQ ID NOS:34-51, 93-98, 100-103 and 123-124, activefragments thereof, or variants thereof can be used as a reporter gene inthe methods provided herein.

Also included in the methods provided herein are variants of any of SEQID NOS: 34-51, 93-98, 100-103 and 123-124 that have at least or about atleast or about or 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or more sequence identity to any of SEQ ID NOS: 34-51, 93-98,100-103 and 123-124. Variants include variants that contain conservativeand non-conservative mutations. It is understood that residues that areimportant or otherwise required for the activity of a luciferase, suchas any described above or known to skill in the art, are generallyinvariant and cannot be changed. Luciferases for use in the methodsprovided herein also can include variants that have an amino acidmodification and that exhibits an altered, such as improved, activitycompared to a luciferase not including the modification. Such variantsinclude those that contain an amino acid modification that enhances thecatalytic activity of the luciferase. For example, the amino acidmodification can be an amino acid replacement (substitution), deletionor insertion.

In the luciferase reaction, light is emitted when luciferase acts on theappropriate luciferin substrate. Photon emission can be detected bylight sensitive apparatus such as a luminometer or modified opticalmicroscopes. Luciferase substrates, e.g., luciferins, are set forth insection b below and Table 3.

iv. Chloramphenicol Acetyltransferases

Chloramphenicol acetyltransferase (CAT) are bacterial enzymes (EC2.3.1.28) that detoxify the antibiotic chloramphenicol and areresponsible for chloramphenicol resistance in bacteria. These enzymescovalently attach an acetyl group from acetyl-CoA to chloramphenicol,resulting in acetylated chloramphenicol. A histidine residue, located inthe C-terminal section of the enzyme, plays a central role in itscatalytic mechanism.

The crystal structure of the type III enzyme from Escherichia coli withchloramphenicol bound has been determined. CAT is a trimer of identicalsubunits (monomer Mr 25,000) and the trimeric structure is stabilized bya number of hydrogen bonds, some of which result in the extension of abeta-sheet across the subunit interface. Chloramphenicol binds in a deeppocket located at the boundary between adjacent subunits of the trimer,such that the majority of residues forming the binding pocket belong toone subunit while the catalytically essential histidine belongs to theadjacent subunit. The active site contains a histidine that isappropriately positioned to act as a general base catalyst in thereaction, and the required tautomeric stabilization is provided by anunusual interaction with a main-chain carbonyl oxygen (Leslie A G (1990)J Mol Biol 213:167-186).

Chloramphenicol acetyltransferases that can be used in the methodsprovided herein include, but are not limited to, those from E. colistrain DJ33-16 (SEQ ID NO:53, DNA set forth in SEQ ID NO:52),Pseudomonas aeruginosa (SEQ ID NO:55, DNA set forth in SEQ ID NO:54),Staphylococcus aureus (SEQ ID NO:57, DNA set forth in SEQ ID NO:56),Agrobacterium tumefaciens (SEQ ID NO:59, DNA set forth in SEQ ID NO:58),Clostridium perfingens (SEQ ID NO:61, DNA set forth in SEQ ID NO:60),Klebsiella pneumoniae (SEQ ID NO:63, DNA set forth in SEQ ID NO:62),Haemophilus influenzae (SEQ ID NO:65, DNA set forth in SEQ ID NO:64),Streptococcus agalactiae (SEQ ID NO:67, DNA set forth in SEQ ID NO:66),Bacillus pumilus (SEQ ID NO:69, DNA set forth in SEQ ID NO:68), Proteusmirabilis (SEQ ID NO:71, DNA set forth in SEQ ID NO:70), Salmonellaenterica (SEQ ID NO:73, DNA set forth in SEQ ID NO:72), Staphylococcusintermedius (SEQ ID NO:75, DNA set forth in SEQ ID NO:74), Listonellaanguillarum (SEQ ID NO:77, DNA set forth in SEQ ID NO:76), Campylobactercoli (SEQ ID NO:79, DNA set forth in SEQ ID NO:78) and Acinetobacterbaumannii (SEQ ID NO:81, DNA set forth in SEQ ID NO:80).

Also included in the methods provided herein are variants of any of SEQID NOS:52-81 that have at least or about at least or about or 60%, 70%,80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequenceidentity to any of SEQ ID NOS: 52-81. Variants include variants thatcontain conservative and non-conservative mutations. It is understoodthat residues that are important or otherwise required for the activityof a chloramphenicol acetyltransferase, such as any described above orknown to skill in the art, are generally invariant and cannot bechanged. Chloramphenicol acetyltransferases for use in the methodsprovided herein also can include variants that have an amino acidmodification and that exhibits an altered, such as improved, activitycompared to a chloramphenicol acetyltransferase not including themodification. Such variants include those that contain an amino acidmodification that enhances the catalytic activity of the chloramphenicolacetyltransferase. For example, the amino acid modification can be anamino acid replacement (substitution), deletion or insertion.Chloramphenicol acetyltransferase substrates include both acetylcoenzyme A (acetyl CoA) and chloramphenicol and are set forth in sectionb below and Table 3.

v. Alkaline Phosphatases

Alkaline phosphatases (ALP, ALKP) (EC 3.1.3.1) are hydrolase enzymesresponsible for removing phosphate groups, e.g., dephosphorylating, frommany types of molecules, including nucleotides, proteins, and alkaloids.Alkaline phosphatases are most effective in an alkaline environment.Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate estersin alkaline buffer and produces an organic radical and inorganicphosphate.

Exemplary alkaline phosphatases include, but are not limited to, shrimpalkaline phosphatase (SAP), from a species of Arctic shrimp (Pandalusborealis) (SEQ ID NO:109, DNA set forth in SEQ ID NO:108), IntestinalAlkaline Phosphatase (AIP; SEQ ID NO:111, DNA set forth in SEQ IDNO:110) and Placental alkaline phosphatase (PALP; SEQ ID NOS:113, DNAset forth in SEQ ID NO:99 and 112) and secreted alkaline phosphatase(SEAP; SEQ ID NO:126), a C terminally truncated version of PALP.

Also included in the methods provided herein are variants of any of SEQID NOS:108-113 and 126 that have at least or about at least or about or60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moresequence identity to any of SEQ ID NOS: 108-113 and 126. Variantsinclude variants that contain conservative and non-conservativemutations. It is understood that residues that are important orotherwise required for the activity of an alkaline phosphatase, such asany described above or known to skill in the art, are generallyinvariant and cannot be changed. Alkaline phosphatases for use in themethods provided herein also can include variants that have an aminoacid modification and that exhibits an altered, such as improved,activity compared to an alkaline phosphatase not including themodification. Such variants include those that contain an amino acidmodification that enhances the catalytic activity of the alkalinephosphatase. For example, the amino acid modification can be an aminoacid replacement (substitution), deletion or insertion. Alkalinephosphatase substrates are set forth in section b below and Table 3.

b. Reporter Enzyme Substrates

A reporter enzyme substrate is any substrate, for example, any compound,that is a substrate for the reporter enzyme. A reporter enzyme iscapable of reacting with the substrate causing a change in the substratethat can be monitored directly or indirectly. Typically, a reporterenzyme substrate is a compound that is modified upon reaction with thereporter enzyme resulting in a modified compound that can be directlymonitored. For example, reporter enzyme substrates typically arecolorless or non-fluorescent substrates or compounds that aretransformed into colored or fluorescent products upon reaction with thereporter enzyme. For example, in the method provided herein, a reporterenzyme substrate can be a fluorescent, fluorogenic, luminescent,luminogenic, chromogenic or spectrophotometric substrate or contrastagent. Reporter enzyme substrates are well known and it is understoodthat a person of skill in the art can select a suitable substrate foruse in the methods provided herein.

Exemplary reporter enzymes include, but are not limited to,β-glucuronidases, β-galactosidases, luciferase, chloramphenicolacetyltransferase (CAT) and alkaline phosphatase. One of skill in theart recognizes each reporter enzyme reacts with a substrate specific tothe reporter enzyme. Exemplary reporter enzymes and their substrates areset forth in Table 3 below. Typically, reporter enzyme substrates usedin the methods provided herein are fluorogenic substrates.

TABLE 3 Exemplary Reporter Enzymes and Substrates Reporter EnzymeSubstrate (Abs/Em of the products) * β-D-Glucuronide Blue-fluorescent4-Methylumbelliferyl-β-D-glucuronide(4- (β-Glucuronidase, product MUG)(360/449) GUS; carboxyumbelliferyl β-D-glucuronide E.C. 3.2.1.31)(CuGlcU) (386/445) Green-fluorescent Fluorescein di-β-D-glucuronide(FDGlcU) product (490/514) 5-(Pentafluorobenzoylamino)fluorescein β-D-glucuronide (PFB-FDGlcU) (490/514) C₁₂-Fluorescein β-D-glucuronide(ImaGene Green ™) (490/514) Chromogenic 5-Bromo-4-chloro-3-indoyl β-D-substrate glucuronide (X-GlcU or BCIG) (615/NA)p-nitrophenyl-β-D-glucuronide phenyl-β-D-glucuronide Red-β-D-GlcU, CHA(Magenta-b-D-GlcA; 5-bromo-6-chloro-3-indolyl-b-D- glucuronide,cyclohexylammmonium salt) Rose-β-D-GlcU, CHA (Salmon-β-D- GlcUA;5-bromo-6-chloro-3-indolyl-β-D- glucuronide, cyclohexylammonium salt)β-D- Blue-fluorescent 4-Methylumbelliferyl β-D- Galactopyranosideproduct galactopyranoside (360/449) (β-Galactosidase, Green-fluorescentFluorescein β-D-galactopyranoside (FDG) E.C. 3.2.1.23) product (490/514)5-(Pentafluorobenzoylamino)-fluorescein β- D-galactopyranoside (490/514)C₂-Fluorescein β-D-galactopyranoside (490/514) C₁₂-Fluoresceinβ-D-galactopyranoside (490/514) 5-Chloromethylfluorescein β-D-galactopyranoside (490/514) Red-fluorescent C₁₂-Resorufin (571/585)product DDAO (646/659) Resorufin (571/585) Chromogenic5-Bromo-4-chloro-3-indoyl-β-D- substrate galactopyranoside (615/NA)Luciferases Luminescent coelenterazine h (E.C. 1.13.12.7) coelenterazinefcp coelenterazine hcp coelenterazine ip coelenterazine f coelenterazinenative D-luciferin ((S)-2-(6-Hydroxy-2-benzothiazolyl)-2-thiazoline-4-carboxylic acid) D-luciferin6′-O-phosphate luciferin 6′-ethyl ether cypridina luciferin(6-(4-Methoxyphenyl)- 2-methyl-3,7-dihydroimidazo[1,2-a]pyrazin-3(7H)-one hydrochloride) click beetle luciferinChloramphenicol Radioactive [¹⁴C]-chloramphenicol acetyltransferases[¹⁴C]-acetyl CoA (E.C. 2.3.1.28) Fluorescent BODIPY ® FL ChloramphenicolBODIPY ® FL 1-deoxychloramphenicol Alkaline Chromogenic p-nitrophenylphosphate (pNPP) (405/NA) phosphatase (ALP, 5-bromo-4-chloro-3-indolylphosphate ALKP, (BCIP) E.C. 3.1.3.1) 5-bromo-4-chloro-3-indolylphosphate/p- iodonitrotetrazolium Violet (BCIP/INT)5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT)5-bromo-4-chloro-3-indolyl phosphate/ tetranitroblue tetrazolium(BCIP/TNBT) 4-Chloro-2-methylbenzenediazonium/3- Hydroxy-2-naphthoicacid 2,4- dimethylanilide phosphate (Fast Red) 3-Hydroxy-2-naphthoicacid 2,4- dimethylanilide phosphate/New Fuchsin 2-hydroxy-3-naphthoicacid anilide phosphate (Naphthol AS phosphate) Fluorescent4-Methylumbelliferyl phosphate disodium salt (MUP) (360 nm/440)fluorescein diphosphate tetraamonium salt (FDP) Ap-New Magenta AP-orangeFirePhos * Approximate absorption (Abs) and fluorescence emission (Em)maxima of enzymatic hydrolysis product, in nm

i. β-Glucuronidase Substrates

An exemplary reporter enzyme for use in the methods herein isβ-glucuronidase. Beta-glucuronidase substrates, include but are notlimited to, fluorescent, spectrophotometric, or chromogenic substratesthat contain the sugar D-glucopyranosiduronic acid attached byglycosidic linkage to a hydroxyl group of a chromogenic, fluorogenic, orother detectable molecule. Fluorescent β-glucuronidase substrates thatcan be used in the methods provided herein include, but are not limitedto, fluorescein di-β-D-glucuronide (FDGlcU),4-methylumbelliferyl-β-D-glucuronide (4-MUG), carboxyumbelliferylβ-D-glucuronide (CUGlcU), 5-(Pentafluorobenzoyl-amino)fluoresceindi-β-D-glucuronide (PFB-FDGlcU), and ImaGene Green™ C₁₂-Fluoresceinβ-D-Glucuronidase Substrate (Invitrogen). Spectrophotometric orchromogenic β-glucuronidase substrates that can be used in the methodsprovided herein, include, but are not limited to,5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GlcU or BCIG),p-nitrophenyl-β-D-glucuronide, Red-β-D-GlcU,CHA (Magenta-b-D-GlcA;5-bromo-6-chloro-3-indolyl-b-D-glucuronide, cyclohexylammonium salt),Rose-β-D-GlcU,CHA (Salmon-β-D-GlcUA;5-bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt),phenyl-β-D-glucuronide, and suitable salts thereof. Exemplaryβ-glucuronidase substrates for use in the methods provided hereininclude fluorescein di-β-D-glucuronide (FDGlcU) and4-methylumbelliferyl-β-D-glucuronide (4-MUG or MUGlcU). It is understoodthat one of skill in the art can select a suitable β-glucuronidasesubstrate for use in the methods provided herein.

ii. β-Galactosidase Substrates

An exemplary reporter enzyme for use in the methods herein isβ-galactosidase. B-Galactosidase substrates, include but are not limitedto, fluorescent, spectrophotometric, or chromogenic substrates thatcontain the sugar D-galactose attached by glycosidic linkage to ahydroxyl group of a chromogenic, fluorogenic, or other detectablemolecule. Fluorescent β-galactosidase substrates that can be used in themethods provided herein include, but are not limited to,4-Methylumbelliferyl 0-D-galactopyranoside, Fluoresceinβ-D-galactopyranoside (FDG), 5-(Pentafluorobenzoylamino)-fluoresceinβ-D-galactopyranoside, C2-Fluorescein β-D-galactopyranoside,C12-Fluorescein β-D-galactopyranoside, 5-Chloromethylfluoresceinβ-D-galactopyranoside, C12-Resorufln, DDAO and Resorufln.Spectrophotometric or chromogenic β-galactosidase substrates that can beused in the methods provided herein, include, but are not limited to,5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal, BCIG) andsuitable salts thereof. It is understood that one of skill in the artcan select a suitable β-galactosidase substrate for use in the methodsprovided herein.

iii. Luciferases

Luciferins are a class of small-molecule substrates that are oxidized inthe presence of the enzyme luciferase to produce oxyluciferin and energyin the form of light. Renilla and Gaussia luciferases use coelenterazineas a substrate, whereas firefly luciferases used D-luciferin. Luciferinsthat can be used as enzyme substrates in the methods provided hereininclude, but are not limited to, firefly luciferin, latia luciferin,bacterial luciferin, including Renilla luciferin, coelenterazineluciferin, dinoflagellate luciferin and virgule or cypridina luciferin.For example, luciferins that can be used in the provided methods includecoelenterazine luciferins, such as coelenterazine h, coelenterazine fcp,coelenterazine hcp, coelenterazine ip, coelenterazine f andcoelenterazine native, luciferins, such as D-luciferin((S)-2-(6-Hydroxy-2-benzothiazolyl)-2-thiazoline-4-carboxylic acid),D-luciferin 6′-O-phosphate, and luciferin 6′-ethyl ether, includingsuitable salts, vargulin or cypridina luciferin(6-(4-Methoxyphenyl)-2-methyl-3,7-dihydroimidazo[1,2-a]pyrazin-3(7H)-onehydrochloride), VivoGlo™ (Promega), EnduRen™ (Promega) and click beetleluciferin.

iv. Chloramphenicol Acetyltransferase (CAT)

Chloramphenicol acetyltransferase requires both chloramphenicol andacetyl coenzyme A (Acetyl CoA) for its activity. CAT activity can bemeasured using either radioactive or fluorescent substrates. Radioactivesubstrates that can be used in the methods provided herein include, forexample, [¹⁴C]-chloramphenicol and [¹⁴C]-acetyl CoA. Fluorescentsubstrates that can be used in the methods provided herein include, butare not limited to, BODIPY® FL (borondipyrromethene difluoridefluorophore) chloramphenicol and BODIPY® FL 1-deoxychloramphenicol.Exemplary substrates for chloramphenicol acetyltransferases as used inthe methods provided herein include fluorescent substrates, such asBODIPY® FL chloramphenicol and BODIPY® FL 1-deoxychloramphenicol.

v. Alkaline Phosphatase

Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate estersin alkaline buffer and produces an organic radical and inorganicphosphate. Chromogenic alkaline phosphatase substrates that can be usedin the methods provided herein include, but are not limited to,p-nitrophenyl phosphate (pNPP), VECTOR® Red (Vector Labs), VECTOR® Blue(Vector Labs), 5-bromo-4-chloro-3-indolyl phosphate (BCIP),5-bromo-4-chloro-3-indolylphosphate/p-iodonitrotetrazolium Violet(BCIP/INT), 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium(BCIP/NBT), 5-bromo-4-chloro-3-indolyl phosphate/tetranitrobluetetrazolium (BCIP/TNBT),4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid2,4-dimethylanilide phosphate (Fast Red), 3-Hydroxy-2-naphthoic acid2,4-dimethylanilide phosphate/New Fuchsin, 2-hydroxy-3-naphthoic acidanilide phosphate (Naphthol AS phosphate) and suitable salts thereof.Exemplary fluorescent alkaline phosphatase substrates include4-Methylumbelliferyl phosphate disodium salt (MUP), fluoresceindiphosphate, tetraamonium salt (FDP), Ap-New Magenta, AP-orange,FirePhos and suitable salts thereof

3. Sample

In the methods provided herein, a biological therapeutic, e.g., a virus,bacterium, cell therapy, immunotherapy, adoptive immunotherapy or genetherapy, is administered to a target locus in a subject. The subject canbe a human subject or a non-human subject, including mammals, such as agorilla, chimpanzee, bovine, ovine, horse, swine, goat or ferret, orfowl, such as chicken, a domestic animal such as a feline or canine, ora rodent, such as a rat, mouse, guinea pig, or any human or mammal towhom a biological therapeutic is administered.

In the provided methods, a sample is obtained from the subject to whomthe biological therapeutic was administered. The sample is obtained froma locus in the subject other than the target of the biological therapyor the sample is not the targeted or administered cell. In otherexamples, the sample does not contain the biological therapeutic. Insome examples, the sample is from tumor tissue or cells that are treatedsuch that the biological therapeutic is not present in the sample or arenormalized to eliminate any contribution from the tumor or target cellsor tissue or therapeutic. For example, the sample can be a blood sampleor bone marrow sample from a patient with leukemia, as long as thesample does not contain the biological therapeutic or is normalized toeliminate any contribution from the biological therapeutic. Samples foruse in the methods herein include body fluids, including but not limitedto blood, plasma, serum, lymph, ascetic fluid, bone marrow, cysticfluid, urine, nipple exudates, sweat, tears, saliva, mouth gargle,peritoneal fluid, cerebrospinal fluid (csf), synovial fluid, aqueoushumour, vitreous humour, amniotic fluid, bile, cerumen (earwax),Cowper's fluid (pre-ejaculatory fluid), Chyle, Chyme, female ejaculate,interstitial fluid, lymph fluid, menses, breast milk, mucus (includingsnot and phlegm), pleural fluid, pus, sebum, semen, vaginal lubrication,and feces. In exemplary embodiments, the sample is blood or serum orurine. In some examples, the sample is a tissue sample, such as, forexample, a tumor biopsy or fine needle aspirate. The sample can becollected in any clinically acceptable manner, but must be collectedsuch that expressed enzyme reporter proteins are preserved. For example,blood collection for routine clinical testing is generally carried outby venepuncture and varying amounts of blood ranging from 5 mL to 20 mLare collected in vaccutainer tubes having color-coded stoppers.

One skilled in the art will recognize that the time period for effectivetreatment with the biological therapeutic will vary. For example, thetime period for infection of a virus will vary depending on the virus,the organ(s) or tissue(s), the immunocompetence of the host and dosageof the virus. Such times can be empirically determined if necessary.Generally, accumulation of expressed reporter protein can be determinedat time points from about less than 1 day, about or 1 day to about 2, 3,4, 5, 6 or 7 days, about or 1 week to about 2, 3 or 4 weeks, about or 1month to about 2, 3, 4, 5, 6 months or longer after infection with thevirus. Thus, samples can be collected between or between about or at 12hours to 1 month, 12 hours to 2 weeks, 12 hours to 7 days, 1 day to 7days, 1 day to 5 days, 1 day to 3 days, 1 day to 2 days, 1 week to 4weeks, 1 week to 3 weeks, 1 week to 2 weeks after treatment with thebiological therapy, or can be collected on or on about 12 hours, 1 day,2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days,11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days,19 days, 20 days, 21 days, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeksafter treatment with the biological therapy. In some examples, samplesare collected hours after treatment, for example, at at least, at aboutor at 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours,13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours or 24 hours after treatment. Inother examples, samples are collected days after treatment, for exampleat at least, at about or at 1 day, 2 days, 3 days, 4 days, 5 days, 6days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 daysafter treatment. In some examples, samples are collected at multipletime points, such as at more than one time point, including, forexample, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more time points.

4. Addition of Reporter Protein Substrates

It is understood by one of skill in the art that the amount of reporterprotein substrate added to the sample can be varied based on thereporter protein, the amount of sample to be detected, the incubationtime and the intensity of the generated signal, and that the amount ofreporter protein substrate added can be increased or decreased asnecessary. It is well within the level of skill in the art to select asuitable substrate and amounts thereof.

In some examples, the reporter enzyme substrate is added to the sampleat an amount between about or at 1 pg to 1 mg, or between, for example,1 pg to 1 mg, 1 pg to 1 μg, 1 pg to 1 ng, 1 pg to 500 pg, 50 pg to 1 mg,50 pg to 1 μg, 50 pg to 1 ng, 50 pg to 500 pg, 250 pg to 1 μg, 250 pg to1 ng, 250 pg to 750 pg, 500 pg to 1 mg, 500 pg to 1 μg, 500 pg to 1 ng,1 ng to 1 mg, 1 ng to 500 μg, 1 ng to 1 μg, 1 ng to 500 ng, 250 ng to 1mg, 250 ng to 500 μg, 250 ng to 250 μg, 250 ng to 1 μg, 250 ng to 750ng, 500 ng to 1 mg, 500 ng to 500 μg, 500 ng to 1 μg, 1 μg to 1 mg, 1 μgto 500 μg, 1 μg to 250 μg, 1 μg to 100 μg, 1 μg to 50 μg, 1 μg to 10 μg,10 μg to 1 mg, 10 μg to 500 μg, 10 μg to 250 μg, 10 μg to 100 μg, 10 μgto 50 μg, 25 μg to 500 μg, 25 μg to 250 μg, 25 μg to 100 μg, 50 μg to 1mg, 50 μg to 500 μg, 50 μg to 250 μg, 50 μg to 100 μg, 100 μg to 1 mg,100 μg to 500 μg, 100 μg to 300 μg, 300 μg to 700 μg, 300 μg to 500 μg,500 μg to 1 mg, or is about or at least about or is 1 pg, 5 pg, 10 pg,20 pg, 30 pg, 40 pg, 50 pg, 100 pg, 150 pg, 200 pg, 250 pg, 300 pg, 350pg, 400 pg, 500 pg, 550 pg, 600 pg, 650 pg, 700 pg, 750 pg, 800 pg, 850pg, 900 pg, 950 pg, 1 ng, 10 ng, 20 ng, 30 ng, 40 ng, 50 ng, 100 ng, 150ng, 200 ng, 250 ng, 300 ng, 350 ng, 400 ng, 450 ng, 500 ng, 550 ng, 600ng, 650 ng, 700 ng, 750 ng, 800 ng, 850 ng, 900 ng, 950 ng, 1 μg, 1.25μg, 1.5 μg, 1.75 μg, 2 μg, 2.25 μg, 2.5 μg, 2.75 μg, 3 μg, 3.25 μg, 3.5μg, 3.75 μg, 4 μg, 4.25 μg, 4.5 μg, 4.75 μg, 5 μg, 5.5 μg, 6 μg, 6.5 μg,7 μg, 7.5 μg, 8 μg, 8.5 μg, 9 μg, 9.5 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40μg, 45 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg,250 μg, 500 μg or 1 mg per sample or any such ranges or amounts.

A sample for use in the methods provided herein can be all or part of asample obtained from a subject. For example, a sample tested in themethods provided herein can be the entire sample taken from the subject,or can be a portion of the sample taken from the subject. Typically, thesample tested in the methods provided herein is a portion of the sampletaken from the subject. For example, the sample for use in the methodscan be a volume between 1 μL to 5 mL, typically between 1 μL and 1 mL,for example, from 1 μL to 500 μL, 1 μL to 250 μL, 1 μL to 100 μL, 1 μLto 75 μL, 1 μL, to 50 μL, 1 μL to 25 μL, 1 μL to 20 μL, 1 μL to 15 μL, 1μL to 10 μL, 1 μL to 5 μL, 5 μL to 500 μL, 5 μL to 250 μL, 5 μL, 50 100μL, 5 μL to 50 μL, 5 μL to 25 μL, 5 μL to 15 μL, 5 μL to 10 μL, 10 μL to500 μL, 10 μL to 250 μL, 10 μL to 100 μL, 10 μL to 50 μL, 10 μL to 25μL, 10 μL to 20 μL, 15 μL to 500 μL, 15 μL to 250 μL, 15 μL to 100 μL,15 μL to 75 μL, 15 μL to 50 μL, 15 μL to 25 μL, 20 μL to 500 μL, 20 μLto 250 μL, 20 μL to 200 μL, 20 μL to 100 μL, 20 μL to 50 μL, 50 μL to500 μL, 50 μL to 250 μL, 50 μL to 200 μL, 50 μL to 150 μL, 50 μL to 100μL, 100 μL to 1000 μL, 100 μL to 250 μL, 100 μL to 200 μL, 100 μL to 150μL, 250 μL to 750 μL, or 500 μL to 1 mL, or the sample is at least or isabout or is 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL,11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 25μL, 30 μL, 35 μL, 40 μL, 45 μL, 50 μL, 60 μL, 70 μL, 75 μL, 80 μL, 90μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL, 150 μL, 160 μL, 170 μL, 180μL, 190 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL, 600μL, 700 μL, 800 μL, 900 μL, or 1 mL or more. In an exemplary example, 20μL of sample was used in the method provided herein.

In other examples, the amount of substrate added is varied in relationto the amount of sample. For example, the ratio of substrate to sampleis from 1:1,000,00 to 1:1, such as, for example, 1:1,000,000 to1:100,000; 1:1,000,000 to 1:10,000; 1:1,000,000 to 1:1,000; 1:500,000 to1:100,000; 1:500,000 to 1:50,000; 1:500,000 to 1:10,000; 1:100,000 to10,000; 1:100,000 to 1:1,000; 1:100,000 to 1:500; 1:50,000 to 1:10,000;1:50,000 to 1:1,000; 1:50,000 to 1:500; 1:10,000 to 1:1,000; 1:10,000 to1:500; 1:10,000 to 1:100; 1:10,000 to 1:1; 1:1000 to 1:500; 1:1000 to1:100; 1:1000 to 1:1; 1:500 to 1:100; 1:500 to 1:1, or the ratio ofsubstrate to sample is at least or at least about or is 1:1,000,000,1:500,000, 1:250,000, 1:100,000, 1:75,000, 1:50,000, 1:25,000, 1:10,000,1:5,000, 1:2,500, 1:1,000, 1:900, 1:800, 1:700, 1:600, 1:500, 1:400,1:300, 1:200, 1:100, 1:50, 1:45, 1:40, 1:35, 1:30, 1:25, 1:20, 1:15,1:10, 1:5, 1:4, 1:3, 1:2, 1:1 or less, weight to volume (w/v) of thesubstrate to sample. In an exemplary example, 3.75 μg of reportersubstrate was added to 20 μL of sample.

5. Incubation of the Sample and the Reporter Protein Substrate

In the method in which enzymes are detected, after the substrate isadded to the sample, the sample and the reporter protein substrate areincubated for a sufficient period of time to allow reaction of thereporter protein and the substrate. For example, the sample and thesubstrate can be incubated for minutes, hours or days, depending on thereporter protein and the substrate. The time periods and conditionstherefore, if necessary, can be empirically determined. In one example,the sample and the substrate are incubated for 10, 20, 30, or 60 minutesprior to detection or other such time period. In another example, thesample and the substrate are incubated for 30 minutes or less prior todetection. In yet another example, the sample and the substrate areincubated for more than one hour prior to detection.

In some examples of the methods provided herein, a sufficient period oftime is from or from about 1 minute to 2 hours, 1 minute to 1 hour, 1minute to 30 minutes, 1 minute to 15 minutes, 15 minutes to 2 hours, 15minutes to 1 hour, 15 minutes to 45 minutes, 15 minutes to 30 minutes,30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 2 hours, 30minutes to 1 hour, 1 hour to 24 hours, 1 hour to 18 hours, 1 hour to 12hours, 1 hour to 6 hours, 1 hour to 3 hours, 1 hour to 2 hours, 6 hoursto 24 hours, 6 hours to 18 hours, 6 hours to 12 hours, 12 hours to 24hours, 12 hours to 18 hours, or is at least or is about or is 1 minute,2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 12 hours, 18 hours or 24 hours or more. It is understood thatreaction times can vary based on reporter protein and substrate, andthat one of skill in the art can select a suitable reaction time.

6. Detection of Activated Substrate or Signal

In the method provided herein, after the sample is incubated with thesubstrate, the activated substrate or signal can be detected using anymethod known to one of skill in the art, including, but not limited, tochromogenic, spectrophotometric, fluorimetric, radioactive andluminescent methods of detection. In one example, a chromogenic orspectrophotometric substrate or signal is detected with aspectrophotometer. In another example, a fluorescent product derivedfrom a fluorogenic substrate or signal is detected with a fluorometer orby fluorescence imaging. In another example, a radioactive substrate orsignal is detected by a scintillation counter, scintigraphy, gammacamera, a β+ detector, a γ detector, or a combination thereof. In yetanother example, photon emission, such as that emitted by a luciferase,can be detected by light sensitive apparatus such as a luminometer ormodified optical microscopes. In another example, signal can be detectedwith a Raman spectrometer. In yet other examples, substrate is detectedwhen changes in fluorescent or optical properties, such as wavelengthchanges, intensity changes or changes in absorption, occur uponactivation or cleavage by the reporter protein. In some examples,detection is effected by capturing with an antibody presented on ananoparticle (see, e.g., Wang et al., (2011) Analyst. 136:4295-4300).

It is understood that the above discussion and following discussion areexemplary only, and, that any biological therapeutic and any protein orproduct encoded thereby, particularly enzyme products, is contemplatedherein, as long as the sample is different from the administeredtherapeutic or does not contain the administered therapeutic or, if itdoes, enhanced amounts of production of the product, are assessed. Themethods herein assess and detect replication of and/or expression ofproducts encoded by a biological therapeutic at a locus or in a tissueor sample that is not the target for the therapy. In particular, thebody fluids, such as any known to those of skill in the art or notedherein, is/are sampled, and a product encoded by the biologicaltherapeutic is detected or signal induced thereby is detected.

D. Methods of Preparing Biological Therapeutics Encoding a ReporterProtein

Biological therapeutics can be prepared by any method known to those ofskill in the art or obtained from commercial sources or by methods to bedeveloped. In some embodiments, in which a heterologous encoded productis detected, the biological therapeutic can be modified or selected toencode such product by any of the well known methods. For example, ifthe detected product is a reporter gene product, such as an enzyme, thereporter gene product can be encoded by the biological therapeutic, suchas a therapeutic vector or microorganism. Typically, genes encoding thereporter proteins are contained within vectors, such as prokaryotic andeukaryotic vectors, including viral vectors, mammalian vectors,bacterial vectors, plant vectors and insect vectors. Alternatively, thegene can be contained in artificial chromosomes. Methods for generatingvectors, including cloning, isolating or modifying nucleic acidmolecules are well known to one of skill in the art.

Any method known to those of skill in the art for identification,selection or production of nucleic acids that encode desired genes, suchas reporter proteins, can be used. Any method available in the art canbe used to obtain a full length (i.e., encompassing the entire codingregion) cDNA or genomic DNA clone encoding a reporter protein, such asfrom a cell or tissue source. Methods for amplification of nucleic acidscan be used to isolate nucleic acid molecules encoding a desiredpolypeptide, including for example, polymerase chain reaction (PCR)methods. A nucleic acid containing material can be used as a startingmaterial from which a desired polypeptide-encoding nucleic acid moleculecan be isolated. For example, DNA and mRNA preparations, cell extracts,tissue extracts, fluid samples (e.g. blood, serum, saliva), samples fromhealthy and/or diseased subjects can be used in amplification methods.Nucleic acid libraries also can be used as a source of startingmaterial. Primers can be designed to amplify a gene encoding a desiredpolypeptide. For example, primers can be designed based on expressedsequences from which a desired polypeptide is generated. Primers can bedesigned based on back-translation of a polypeptide amino acid sequence.Nucleic acid molecules generated by amplification can be sequenced andconfirmed to encode a desired polypeptide.

Additional nucleotide sequences can be joined to a polypeptide-encodingnucleic acid molecule, including linker sequences containing restrictionendonuclease sites for the purpose of cloning the synthetic gene into avector, for example, a protein expression vector or a vector designedfor the amplification of the core protein coding DNA sequences.Furthermore, additional nucleotide sequences specifying functional DNAelements can be operatively linked to a polypeptide-encoding nucleicacid molecule. Examples of such sequences include, but are not limitedto, promoter sequences designed to facilitate intracellular proteinexpression, and secretion sequences, for example heterologous signalsequences, designed to facilitate protein secretion. Such sequences areknown to those of skill in the art.

The identified and isolated nucleic acids then can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art can be used. Possible vectors include, but are not limitedto, plasmids or modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pCMV4, pBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene, La Jolla, Calif.). The insertion into a cloning vector can,for example, be accomplished by ligating the DNA fragment into a cloningvector which has complementary cohesive termini. Insertion can beeffected using TOPO cloning vectors (Invitrogen, Carlsbad, Calif.). Ifthe complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules can beenzymatically modified. Alternatively, any site desired can be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers can contain specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and protein genecan be modified by homopolymeric tailing. Recombinant molecules can beintroduced into host cells via, for example, transformation,transfection, infection, electroporation and sonoporation, so that manycopies of the gene sequence are generated.

1. Vectors

For generation of a vector containing a gene encoding a reporter proteinfor use in a biological therapeutic to be detected by the methodsprovided herein, the nucleic acid containing all or a portion of thenucleotide sequence encoding the reporter protein can be inserted intoan appropriate expression vector, i.e., a vector that contains thenecessary elements for the transcription and translation of the insertedprotein coding sequence. The necessary transcriptional and translationalsignals also can be supplied by the native promoter for enzyme genes,and/or their flanking regions. Provided are vectors that contain asequence of nucleotides that encodes a reporter protein, such as aβ-glucuronidase, coupled to the native or heterologous signal sequence,as well as multiple copies thereof. The vectors can be selected forexpression of the protein in the cell or such that the protein isexpressed as a secreted protein.

A variety of host-vector systems can be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus and otherviruses); insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements can be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector can be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods can include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding protein, or domains,derivatives, fragments or homologs thereof, can be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins can be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for a desired protein. Promoterswhich can be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al. Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA78:5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25 (1983)); see also “Useful Proteins from Recombinant Bacteria”:in Scientific American 242:79-94 (1980)); plant expression vectorscontaining the nopaline synthetase promoter (Herrera-Estrella et al.,Nature 303:209-213 (1984)) or the cauliflower mosaic virus 35S RNApromoter (Gardner et al., Nucleic Acids Res. 9:2871 (1981)), and thepromoter of the photosynthetic enzyme ribulose bisphosphate carboxylase(Herrera-Estrella et al., Nature 310:115-120 (1984)); promoter elementsfrom yeast and other fungi such as the Gal4 promoter, the alcoholdehydrogenase promoter, the phosphoglycerol kinase promoter, thealkaline phosphatase promoter, and the following animal transcriptionalcontrol regions that exhibit tissue specificity and have been used intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., Cell 38:639-646 (1984); Ornitz etal., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986); MacDonald,Hepatology 7:42S-51S (1987)); insulin gene control region which isactive in pancreatic beta cells (Hanahan et al., Nature 315:115-122(1985)), immunoglobulin gene control region which is active in lymphoidcells (Grosschedl et al., Cell 38:647-658 (1984); Adams et al., Nature318:533-538 (1985); Alexander et al., Mol. Cell Biol. 7:1436-1444(1987)), mouse mammary tumor virus control region which is active intesticular, breast, lymphoid and mast cells (Leder et al., Cell45:485-495 (1986)), albumin gene control region which is active in liver(Pinkert et al., Genes and Devel. 1:268-276 (1987)), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., Mol.Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science 235:53-58 1987)),alpha-1 antitrypsin gene control region which is active in liver (Kelseyet al., Genes and Devel. 1:161-171 (1987)), beta globin gene controlregion which is active in myeloid cells (Magram et al., Nature315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)), myelin basicprotein gene control region which is active in oligodendrocyte cells ofthe brain (Readhead et al., Cell 48:703-712 (1987)), myosin lightchain-2 gene control region which is active in skeletal muscle (Shani,Nature 314:283-286 (1985)), and gonadotrophic releasing hormone genecontrol region which is active in gonadotrophs of the hypothalamus(Mason et al., Science 234:1372-1378 (1986)).

For example, a vector is used that contains a promoter operably linkedto nucleic acids encoding a reporter protein, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pQEexpression vectors (available from Qiagen, Valencia, Calif.; see alsoliterature published by Qiagen describing the system). pQE vectors havea phage T5 promoter (recognized by E. coli RNA polymerase) and a doublelac operator repression module to provide tightly regulated, high-levelexpression of recombinant proteins in E. coli, a synthetic ribosomalbinding site (RBS II) for efficient translation, a 6XHis tag codingsequence, t₀ and T1 transcriptional terminators, ColE1 origin ofreplication, and a beta-lactamase gene for conferring ampicillinresistance. The pQE vectors enable placement of a 6xHis tag at eitherthe N- or C-terminus of the recombinant protein. Such plasmids includepQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for allthree reading frames and provide for the expression of N-terminally6xHis-tagged proteins. Other exemplary plasmid vectors fortransformation of E. coli cells, include, for example, the pETexpression vectors (see, U.S. Pat. No. 4,952,496; available fromNovagen, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a-c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET 19b (Novagen, Madison, Wis.), which contain a His-Tag™ leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn, the T7-lac promoter region and the T7 terminator.

In other embodiments, organ or tissue-specific expression of a reporterprotein within a biological therapeutic can be controlled by regulatorysequences. In order to achieve expression only in the target organ, forexample, a tumor to be treated, the foreign nucleotide sequence can belinked to a tissue specific promoter and used for gene therapy. Suchpromoters are well known to those skilled in the art (see e.g.,Zimmermann et al., (1994) Neuron 12, 11-24; Vidal et al.; (1990) EMBO J.9, 833-840; Mayford et al., (1995), Cell 81, 891-904; Pinkert et al.,(1987) Genes & Dev. 1, 268-76).

2. Viruses

The viruses for use in the methods provided herein can be formed bystandard methodologies well known in the art for producing and/ormodifying viruses. Briefly, the methods can include introducing intoviruses one or more genetic modifications, followed by screening theviruses for properties reflective of the modification or for otherdesired properties.

a. Genetic Modifications

Standard techniques in molecular biology can be used to generate themodified viruses for use in the methods provided herein. Such techniquesinclude various nucleic acid manipulation techniques, nucleic acidtransfer protocols, nucleic acid amplification protocols, and othermolecular biology techniques known in the art. For example, pointmutations can be introduced into a gene of interest through the use ofoligonucleotide mediated site-directed mutagenesis. Alternatively,homologous recombination can be used to introduce a mutation orexogenous sequence into a target sequence of interest. In an alternativemutagenesis protocol, point mutations in a particular gene also can beselected for using a positive selection pressure. See, e.g., CurrentTechniques in Molecular Biology, (Ed. Ausubel, et al.). Nucleic acidamplification protocols include but are not limited to the polymerasechain reaction (PCR). Use of nucleic acid tools such as plasmids,vectors, promoters and other regulating sequences, are well known in theart for a large variety of viruses and cellular organisms. Nucleic acidtransfer protocols include calcium chloride transformation/transfection,electroporation, liposome mediated nucleic acid transfer,N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfatemediated transformation, and others. Further a large variety of nucleicacid tools are available from many different sources including ATCC, andvarious commercial sources. One skilled in the art will be readily ableto select the appropriate tools and methods for genetic modifications ofany particular virus according to the knowledge in the art and designchoice.

Any of a variety of modifications can be readily accomplished usingstandard molecular biological methods known in the art. Themodifications will typically be one or more truncations, deletions,mutations or insertions of the viral genome. In one embodiment, themodification can be specifically directed to a particular sequence. Themodifications can be directed to any of a variety of regions of theviral genome, including, but not limited to, a regulatory sequence, to agene-encoding sequence, or to a sequence without a known role. Any of avariety of regions of viral genomes that are available for modificationare readily known in the art for many viruses, including the virusesspecifically listed herein. As a non-limiting example, the loci of avariety of vaccinia genes provided herein and elsewhere exemplify thenumber of different regions that can be targeted for modification in theviruses provided herein. In another embodiment, the modification can befully or partially random, whereupon selection of any particularmodified virus can be determined according to the desired properties ofthe modified the virus. These methods include, for example, in vitrorecombination techniques, synthetic methods and in vivo recombinationmethods as described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor LaboratoryPress, cold Spring Harbor NY (1989), and in the Examples disclosedherein.

The viruses for use as therapeutics detected by the methods providedherein encode a reporter protein, for example, an enzyme, including forexample, β-glucuronidase, β-galactosidase, luciferases, chloramphenicolacetyltransferases or alkaline phosphatase. In some embodiments, thevirus can be modified to express an additional exogenous gene. Exemplaryexogenous gene products include proteins and RNA molecules. The modifiedviruses can express a detectable gene product, a therapeutic geneproduct, a gene product for manufacturing or harvesting, or an antigenicgene product for antibody harvesting. The characteristics of such geneproducts are described herein and elsewhere. In some embodiments ofmodifying an organism to express an exogenous gene, the modification canalso contain one or more regulatory sequences to regulate expression ofthe exogenous gene. As is known in the art, regulatory sequences canpermit constitutive expression of the exogenous gene or can permitinducible expression of the exogenous gene. Further, the regulatorysequence can permit control of the level of expression of the exogenousgene. In some examples, inducible expression can be under the control ofcellular or other factors present in a tumor cell or present in avirus-infected tumor cell. In other examples, inducible expression canbe under the control of an administrable substance, including IPTG,RU486 or other known induction compounds. Any of a variety of regulatorysequences are available to one skilled in the art and can be selectedaccording to known factors and design preferences. In some embodiments,such as gene product manufacture and harvesting, the regulatory sequencecan result in constitutive, high levels of gene expression. In someembodiments, such as anti-(gene product) antibody harvesting, theregulatory sequence can result in constitutive, lower levels of geneexpression. In tumor therapy embodiments, a therapeutic protein can beunder the control of an internally inducible promoter or an externallyinducible promoter.

In other embodiments, organ or tissue-specific expression can becontrolled by regulatory sequences. In order to achieve expression onlyin the target organ, for example, a tumor to be treated, the foreignnucleotide sequence can be linked to a tissue specific promoter and usedfor gene therapy. Such promoters are well known to those skilled in theart (see e.g., Zimmermann et al., Neuron 12: 11-24 (1994); Vidal et al.,EMBO J. 9: 833-840 (1990); Mayford et al., Cell 81: 891-904 (1995); andPinkert et al., Genes & Dev. 1: 268-76 (1987)).

In some embodiments, the viruses can be modified to express two or moreproteins, where any combination of the two or more proteins can be oneor more detectable gene products, therapeutic gene products, geneproducts for manufacturing or harvesting or antigenic gene products forantibody harvesting. In one embodiment, a virus can be modified toexpress a detectable protein and a therapeutic protein. In anotherembodiment, a virus can be modified to express two or more gene productsfor detection or two or more therapeutic gene products. For example, oneor more proteins involved in biosynthesis of a luciferase substrate canbe expressed along with luciferase. When two or more exogenous genes areintroduced, the genes can be regulated under the same or differentregulatory sequences, and the genes can be inserted in the same ordifferent regions of the viral genome, in a single or a plurality ofgenetic manipulation steps. In some embodiments, one gene, such as agene encoding a detectable gene product, can be under the control of aconstitutive promoter, while a second gene, such as a gene encoding atherapeutic gene product, can be under the control of an induciblepromoter. Methods for inserting two or more genes into a virus are knownin the art and can be readily performed for a wide variety of virusesusing a wide variety of exogenous genes, regulatory sequences, and/orother nucleic acid sequences.

Methods of producing recombinant viruses are known in the art. Providedherein for exemplary purposes are methods of producing a recombinantvaccinia virus. A recombinant vaccinia virus with an insertion in theF14.5L gene (NotI site of LIVP) can be prepared by the following steps:(a) generating (i) a vaccinia shuttle plasmid containing the modifiedF14.5L gene inserted at restriction site X and (ii) a dephosphorylatedwt VV (VGL) DNA digested at restriction site X; (b) transfecting hostcells infected with PUV-inactivated helper VV (VGL) with a mixture ofthe constructs of (i) and (ii) of step a; and (c) isolating therecombinant vaccinia viruses from the transfectants. One skilled in theart knows how to perform such methods, for example by following theinstructions given in U.S. Publication Nos. 2005-0031643 and 2006-051370and U.S. Pat. Nos. 7,588,767 and 7,588,771; see also Timiryasova et al.(Biotechniques 31: 534-540 (2001)). In one embodiment, restriction siteX is a unique restriction site. A variety of suitable host cells alsoare known to the person skilled in the art and include many mammalian,avian and insect cells and tissues which are susceptible for vacciniavirus infection, including chicken embryo, rabbit, hamster and monkeykidney cells, for example, HeLa cells, RK₁₃, CV-1, Vero, BSC40 and BSC-1monkey kidney cells.

b. Control of Heterologous Gene Expression

In some embodiments, expression the therapeutic product can becontrolled by a regulatory sequence. Suitable regulatory sequenceswhich, for example, are functional in a mammalian host cell are wellknown in the art. In one example, the regulatory sequence contains apoxvirus promoter. In another embodiment, the regulatory sequence cancontain a natural or synthetic vaccinia virus promoter. Strong latepromoters can be used to achieve high levels of expression of theforeign genes. Early and intermediate-stage promoters also can be used.In one embodiment, the promoters contain early and late promoterelements, for example, the vaccinia virus early/late promoter P7.5k,vaccinia late promoter P11k, a synthetic early/late vaccinia PSELpromoter (Patel et al., (1988) Proc. Natl. Acad. Sci. USA 85: 9431-9435;Davison and Moss, (1989) J Mol Biol 210: 749-769; Davison et al. (1990)Nucleic Acids Res. 18: 4285-4286; Chakrabarti et al. (1997),BioTechniques 23: 1094-1097). As described elsewhere herein, the virusesprovided can exhibit differences in characteristics, such asattenuation, as a result of using a stronger promoter versus a weakerpromoter. For example, in vaccinia, synthetic early/late and latepromoters are relatively strong promoters, whereas vaccinia syntheticearly, P7.5k early/late, P7.5k early, and P28 late promoters arerelatively weaker promoters (see e.g., Chakrabarti et al. (1997)BioTechniques 23(6) 1094-1097). Combinations of different promoters canbe used to express different gene products in the same virus or twodifferent viruses. In one embodiment, different therapeutic ordetectable gene products are expressed from different promoters, such astwo different vaccinia synthetic promoters.

3. Bacteria

The bacteria used in the methods provided herein can be formed bystandard methodologies well known in the art for producing or modifyingbacteria. Briefly, the methods include introducing into the bacteria oneor more genetic modification(s), followed by screening the bacteria forproperties reflective of the modification(s) or for other desiredproperties. Exemplary nucleic acid molecular modifications includetruncations, insertions, deletions and mutations. In an exemplarymodification, bacteria can be modified by truncation, insertion,deletion or mutation of one or more genes. In some examples, nucleicacid carrying multiple genes can be inserted into the genome of thebacterium or provided on a plasmid. The bacteria for use in the methodsprovided herein are modified to encode a reporter gene, including forexample, β-glucuronidase, β-galactosidase, luciferases, chloramphenicolacetyltransferases or alkaline phosphatase. In one example, a bacteriumcan be modified to carry the lux operon for the production of bacterialluciferase and proteins for the generation of the bacterial luciferasesubstrate. In an exemplary modification, an endogenous gene, anexogenous gene or a combination thereof can be inserted into a plasmidwhich is inserted into the bacteria using any of the methods known inthe art. Methods for optimizing expression genes are known in the artand include, for example, modification of copy number, promoterstrength, deletion of genes that encode inhibitory proteins, or movementof essential genes to a plasmid in order to maintain the plasmid in thetransformed bacteria. The modifications can be directed to any of avariety of regions of the bacterial genome or endogenous plasmids,including, but not limited to, a regulatory sequence, to a gene-encodingsequence, or to a sequence without a known role. Any of a variety ofregions of bacterial genomes that are available for modification arereadily known in the art for many bacteria, including the bacteriaspecifically listed herein.

Standard techniques in molecular biology can be used to generate themodified bacteria for use in the methods provided. Such techniquesinclude various nucleic acid manipulation techniques, nucleic acidtransfer protocols, nucleic acid amplification protocols, and othermolecular biology techniques known in the art. For example, pointmutations can be introduced into a gene of interest through the use ofoligonucleotide mediated site-directed mutagenesis. Alternatively,homologous recombination techniques can be used to introduce a mutationor exogenous sequence into a target sequence of interest; or can be usedto inactivate a target sequence of interest. Nucleic acid transferprotocols include calcium chloride transformation/transfection,transduction, electroporation, liposome mediated nucleic acid transferand others. In an alternative mutagenesis protocol, point mutations in aparticular gene also can be selected for using a positive selectionpressure. See, e.g., Current Protocols in Molecular Biology, (ed.Ausubel, et al.). Nucleic acid amplification protocols include but arenot limited to the polymerase chain reaction (PCR). Use of nucleic acidtools such as plasmids, vectors, promoters and other regulatingsequences, are well known in the art for a large variety of organismsfor use in bacterial expression systems. Plasmids can be created tocarry genes using methods known to one skilled in the art. High copyplasmids can be used to cause over-expression of endogenous orheterologous proteins in a bacterium. Further, a large variety ofnucleic acid tools are available from many different sources includingthe American Type Culture Collection (ATCC), and various commercialsources. One skilled in the art will be readily able to select theappropriate tools and methods for genetic modifications of anyparticular bacterium according to the knowledge in the art and designchoice.

Expression of exogenous genes can be controlled by a constitutivepromoter, or by an inducible promoter. Expression also can be influencedby one or more proteins or RNA molecules expressed by the bacteria.Genes can be encoded in a bacterial chromosome or on a plasmid.Over-expression of a gene or gene product can be achieved by insertionof a gene into the bacterial chromosome under the control of a strongpromoter. Plasmids can be created to carry genes using methods known toone skilled in the art. High copy plasmids can be used to causeover-expression of exogenous proteins in bacteria. Plasmids forexpression of proteins include, but are not limited to ColE1, pBR322,p15A, pEMBLex2, pMAL-p2, pUC18A2 (a pUC18-derived plasmid containing theftn gene), pUC118, pGS281, pMK4, pUNK1, pAMβ1 and pTA1060. Choice of aplasmid for expression at desired levels is well-known in the art aswell as techniques to introduce genes into the plasmids (Sambrook et al.Molecular Cloning: A Laboratory Manual. 2^(nd) ed. Cold Spring HarborLaboratory Press, New York, N.Y. 1989; Current Protocols in MolecularBiology. Ed. Ausubel et al. John Wiley & Sons, Inc. Cambridge, Mass.,1995).

E. Methods for Detecting and Monitoring Therapy

Methods for detecting, assessing and/or monitoring therapy by abiological therapeutic are provided. The methods can detect and/ormonitor the effectiveness of therapy. For example, the methods fordetecting colonization or replication of or by a biological therapeuticcan be used, for example, for detecting and/or diagnosing diseases anddisorders, evaluating the efficacy or progress of a treatment or therapyfor a disease or disorder, evaluating or determining an optimal time ofinduction of therapeutic gene expression for a bacterial- orviral-mediated treatment or therapy for a disease or disorder,developing non-human animal models for diseases and disorders, assayingor screening compositions for potential use as therapeutic agents forthe treatment of diseases and disorders and for tracking or monitoringdelivery of compositions to cells and tissues, including, sites ofcellular proliferation, tumors, tumor tissues, tumor cells, includingcirculating tumor cells, metastases, areas of inflammation, wounds andinfections.

The methods for detecting colonization or replication of a biologicaltherapeutic provided herein, such as an oncolytic virus or adoptiveimmunotherapy, can be used to monitor treatment of cancers and tumors,such as, but not limited to, bladder tumors, breast tumors, prostatetumors, glioma tumors, adenocarcinomas; ovarian carcinomas, andpancreatic carcinomas, liver tumors and skin tumors, pancreatic cancer,non-small cell lung cancer, multiple myeloma, or leukemia;cancer-forming solid tumors, such as lung and bronchus, breast, colonand rectum, kidney, stomach, esophagus, liver and intrahepatic bileduct, urinary bladder, brain and other nervous system, head and neck,oral cavity and pharynx, cervix, uterine corpus, thyroid, ovary, testes,prostate, malignant melanoma, cholangiocarcinoma, thymoma, non-melanomaskin cancers, as well as hematologic tumors and/or malignancies, such aschildhood leukemia and lymphomas, multiple myeloma, Hodgkin's disease,lymphomas of lymphocytic and cutaneous origin, acute and chronicleukemia such as acute lymphoblastic, acute myelocytic or chronicmyelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancersassociated with AIDS. In addition, treatment of other metastaticdiseases can be monitored by the methods provided herein.

The methods for detecting colonization or replication of a biologicaltherapeutic provided herein, such as an oncolytic virus or adoptiveimmunotherapy, can be used to detect the presence of tumors or tumorcells, including circulating tumor cells and metastasizing cells.Metastasis involves the formation of progressively growing tumor foci atsites secondary to a primary lesion (Yoshida et al. (2000) J. Natl.Cancer Inst. 92(21):1717-1730; Welch et al. (1999) J. Natl. Cancer Inst.91:1351-1353) and is a major cause of morbidity and mortality in humanmalignancies (Nathoo et al. J. Clin. Pathol. 58:237-242 (2005); Fidleret al. Cell 79:185-188 (1994)). In vivo metastasis follows a series ofsteps known as the metastatic cascade, in which tumor cells invade localtissue, intravasate through the bloodstream or lymphatics as emboli orsingle tumor cells (i.e. circulating tumor cells (CTCs)), and aretransported to secondary sites, where they can lodge into themicrovasculature and form metastatic lesions (Kauffman et al. J. Urology169:1122-1133 (2003).

Circulating tumor cells were first observed in blood samples of deceasedpatients with advanced cancers as early as 1869 (Ashworth (1869) AustMed J 14:146-149). More recently, studies on clinical samples,particularly in breast, colon and prostate cancer patients, have shown acorrelation between the presence of CTCs in the peripheral blood andcancer prognosis. Detection of CTCs is predictive of metastatic disease,and the quantity of CTCs detected correlates with the severity ofmetastatic disease. The presence of CTCs in patient samples aftertherapy also has been associated with tumor progression and spread, poorresponse to therapy, relapse of disease, and/or decreased survival overa period of several years. Detection of CTCs can provide a means forearly detection and treatment of metastatic disease and monitoring ofdisease therapy.

Because circulating tumor cells (CTCs) have the potential to form tumorsand their quantity in circulation correlates with metastatic disease,the ability to accurately identify and quantify CTCs in patient sampleswould aid in the early diagnosis and prognosis of many types of cancersand the monitoring of cancer treatments. Effective detection of CTCs inbodily samples, such as in the blood, lymph or other bodily fluids, alsowould aid in staging of particular tumors and evaluating metastaticactivity.

Detection of proteins produced by and, secreted by cells containing, thebiological therapeutic can be used as a simple marker to indicate cellsurvival. Genetically altered cells secrete active enzyme as long asthey are viable. A small number of cells is sufficient for detection andthe amount of reporter protein in the blood correlates with the amountof cells producing the enzyme. Therefore, cell therapies and tissueregeneration are monitored by the methods herein. The detection of cellsurface associated reporter protein can aid in studies relying ontransfection of cells or following bacteria or parasite infections inwhich blood-borne pathogens express a membrane- or cell-wall-anchoredreporter protein. Also, non-membrane passing prodrug therapies benefitfrom detection of a reporter protein, such as β-glucuronidase in theblood as the reporter protein is only observed upon successful prodrugtreatment as only then, the active protein is released from the tumor.Other benefits and applications of the methods provided herein will beapparent to the skilled artisan and are contemplated herein.

A tumor or metastasis can be detected by physical examination ofsubject, laboratory tests, such as blood or urine tests, imaging andgenetic testing, such as testing for gene mutations that are known tocause cancer. For example, a tumor or metastasis can be detected usingin vivo imaging techniques, such as digital X-ray radiography,mammography, CT (computerized tomography) scanning, MRI (magneticresonance imaging), ultrasonography and PET (positron emissiontomography) scanning. Alternatively, a tumor can be detected using tumormarkers in blood, serum or urine, that is by monitoring substancesproduced by tumor cells or by other cells in the body in response tocancer. For example, prostate specific antigen (PSA) levels are used todetect prostate cancer in men. Additionally, tumors can be detected andmonitored by biopsy.

The effectiveness of a biological therapy for treatment of a tumor canbe externally monitored (e.g., external measurement of tumor size) or bymonitoring the animal (e.g., monitoring animal weight, blood panel,antibody titer, spleen size, or liver size). Any of a variety ofmonitoring steps can be used to monitor a biological therapeutic,including, but not limited to, monitoring tumor size, monitoringanti-(tumor antigen) antibody titer, monitoring the presence and/or sizeof metastases, monitoring the subject's lymph nodes, monitoring thesubject's weight or other health indicators including blood or urinemarkers, monitoring anti-(microorganism antigen) antibody titer,monitoring expression of a detectable gene product, and directlymonitoring titer of a microorganism, such as a virus, in a tumor, tissueor organ of a subject.

F. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Vaccinia viruses A. GLV-1h68

The attenuated vaccinia virus strain GLV-1h68 (SEQ ID NO:90), encodingβ-galactosidase and β-glucuronidase, was purified as previouslydescribed (Zhang et al., (2007) Cancer Res 67:10038-10046). Thisgenetically engineered strain, which has been described in U.S. PatentPublication No. 2005/0031643, contains DNA insertions in the F14.5L,thymidine kinase (TK) and hemagglutinin (HA) genes. GLV-1h68 wasprepared from the vaccinia virus strain designated LIVP (a vacciniavirus strain, originally derived by adapting the vaccinia Lister strain(ATCC Catalog No. VR-1549) to calf skin (Research Institute of ViralPreparations, Moscow, Russia, Al'tshtein et al. (1983) Dokl. Akad. NaukUSSR 285:696-699). The LIVP strain, whose genome sequence is set forthin SEQ ID NO: 91 and from which GLV-1h68 was generated, contains amutation in the coding sequence of the TK gene, in which a substitutionof a guanine nucleotide with a thymidine nucleotide (nucleotide position80207 of SEQ ID NO: 91) introduces a premature STOP codon within thecoding sequence.

As described in U.S. Patent Publication No. 2005/0031643 (seeparticularly, Example 1 of the application), GLV-1h68 was generated byinserting expression cassettes encoding detectable marker proteins intothe F14.5L (also designated in LIVP as F3) gene, thymidine kinase (TK)gene, and hemagglutinin (HA) gene loci of the vaccinia virus LIVPstrain. Specifically, an expression cassette containing a Ruc-GFP cDNA(a fusion of DNA encoding Renilla luciferase and DNA encoding GFP; SEQID NO:92) under the control of a vaccinia synthetic early/late promoterP_(SEL) was inserted into the F14.5L gene; an expression cassettecontaining DNA encoding beta-galactosidase (vector psC65; SEQ ID NO:106)under the control of the vaccinia early/late promoter P_(7.5k) (denoted(P_(7.5k))LacZ) and DNA encoding a rat transferrin receptor positionedin the reverse orientation for transcription relative to the vacciniasynthetic early/late promoter P_(SEL) (denoted (P_(SEL))rTrfR) wasinserted into the TK gene (the resulting virus does not expresstransferrin receptor protein since the DNA encoding the protein ispositioned in the reverse orientation for transcription relative to thepromoter in the cassette); and an expression cassette containing DNAencoding β-glucuronidase (pLacGus Plasmid; SEQ ID NO:107) under thecontrol of the vaccinia late promoter P_(11k) (denoted (P_(11k))gusA)was inserted into the HA gene. Insertion of the expression cassettesinto the LIVP genome to generate the GLV-1h68 strain resulted indisruption of the coding sequences for each of the F14.5L, TK and HAgenes; accordingly, all three genes in the resulting strains arenonfunctional in that they do not encode the corresponding full-lengthproteins.

An additional gusA encoding virus used was GLV-1h80, a derivative ofGLV-1h68, in which the lacZ gene was replaced by the murine MCP-1 geneunder control of the promoter P_(SL) (see Table 4 below).

Recombinant viruses were generated by transformation of shuttle plasmidvectors using the FuGENE 6 transfection reagent (Roche Applied Science)into CV-1 cells, which were preinfected with the LIVP parental virus orone of its mutant derivatives. The expression of RUC-GFP fusion proteinby the recombinant viruses was confirmed by luminescence assay andfluorescence microscopy. Expressions of β-galactosidase andβ-glucuronidase A were confirmed by blue plaque formation upon additionof 5-bromo-4-chloro-3-indolyl-h-D-galactopyranoside (X-gal, Stratagene)and 5-bromo-4-chloro-3-indolyl-h-D-glucuronic acid (X-GlcA, ResearchProduct International Corporation), respectively. Positive plaquesformed by the recombinant virus were isolated and purified. The clonalpurity each mutant virus isolate was verified by expression of thecorresponding marker gene(s) in the F14.5L, J2R, and A56R loci, whichwas also confirmed by PCR and DNA sequencing. Viruses were propagated inCV-1 cells, and up to 7×10⁹ plaque-forming unit (pfu)/mL of GLV-1h68 canbe purified from 2×10⁸ infected CV-1 cells through sucrose gradients(Joklik W K (1962) Virology 18:9-18).

B. Generation of Control Viruses rVACV-LacZ and rVACV-gusA⁻

For generation of control viruses, lacZ and gusA of GLV-1h68 werereplaced by nonrelevant gene constructs to create viruses negative forbeta-galactosidase (rVACV-LacZ⁻) and beta-glucuronidase (rVACV-gusA⁻)respectively. The rVACV-LacZ⁻ virus strain was GLV-1h143, in which thelacZ gene was replaced by the human Cyp11B2 gene (see, U.S. Pat. Pub.No. 2009/0117034). The rVACV-GusA⁻ virus strains that were used wereeither GLV-1h90 (gusA replaced by Hyper-IL-6 encoding gene; see, U.S.Pat. Pub. Nos. 2009/0098529 and 2009/0053244) or GLV-1h189 (gusAreplaced by gene encoding the TurboFP635 encoding gene). Viruses werepropagated in CV-1 cells were purified through sucrose gradients (JoklikW K (1962) Virology 18:9-18).

TABLE 4 Generation of engineered vaccinia viruses. Parental Name ofVirus Virus Genotype GLV-1h68 — F14.5L: (P_(SEL))Ruc-GFP TK:(P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(11k))gusA GLV-1h80 GLV-1h68F14.5L: (P_(SEL))Ruc-GFP TK: (P_(SLL))mMCP-1 HA: (P_(11k))gusA GLV-1h90GLV-1h68 F14.5L: (P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA:(P_(SE))sIl-6R/IL-6 GLV-1h143 GLV-1h68 F14.5L: (P_(SEL))Ruc-GFP TK:(P_(SE))CYP11B2 HA: (P_(11k))gusA GLV-1h189 GLV-1h68 F14.5L:(P_(SEL))Ruc-GFP TK: (P_(SEL))rTrfR-(P_(7.5k))LacZ HA: (P_(SEL))FUKW

C. Activity

Heterologous gene expression of the described vaccinia virus strain wasconfirmed by Western blot analysis as well as immuno-staining studies incell culture and infected tumor sections, as described in Example 2below. The assay was tested with purified enzyme as well as with samplesfrom vaccinia virus injected animals. Marker gene expression ofβ-galactosidase, β-glucuronidase and Ruc-GFP in cell culture wasdetermined by Western Blot analysis at 6, 12, 24 and 48 hours post A549cell infection (multiplicity of infection (MOI) of 0.5), with expressionof β-galactosidase observed at 12, 24 and 48 hours for GLV-1h68 andrVACV-gusA⁻ and β-glucoronidase observed at 12, 24 and 48 hours forGLV-1h68 and rVACV-LazZ⁻. GLV-1h68 encodes both beta-galactosidase andbeta-glucuronidase, while rVACV-LacZ⁻ only encodes beta-glucuronidaseand rVACV-gusA⁻ only encodes beta-galactosidase. In viral plaques,GLV-1h68 and rVACV-gusA⁻ were positive for beta-galactosidase activityand GLV-1h68 and rVACV-LazZ⁻ were positive for beta-glucuronidaseactivity.

Example 2 Materials and Methods

In this example, various materials and methods are described.

Cell Culture

Human A549 lung cancer cells (ATCC No. CCL-185) were cultured inRPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1%antibiotic-antimycotic solution (PAA Laboratories, Cölbe, Germany) understandard cell culture conditions (37° C., 5% CO₂). MTH52c, derived froma malignant small-cell canine carcinoma of the mammary gland (Sterenczaket al., (2009) Gene 434:35-42), was cultured in DMEM supplemented withantibiotic-antimycotic solution and 20% FBS.

Infection of Cell Cultures

Two days before infection, cells were seeded in 6-well plates forwestern blot analysis or 12-well plates containing sterile cover slipsfor microscopy studies. 90% confluent cell layers were either mocktreated or infected with GLV-1h68, rVACV-LacZ⁻ or rVACV-GusA⁻ (describedin Example 1 above) at a multiplicity of infection (MOI) of 0.1 for 1 hat 37° C. and 5% CO2 in medium containing 2% FBS. Afterwards theinfection medium was aspirated and replaced by standard cell culturemedium.

Western Blot

For detection of proteins, infected cells were harvested and lysed inSDS sample buffer at 6, 12, 24 and 48 hours post-infection (hpi).Lysates were separated by 10% SDS-polyacrylamide gel electrophoresis andsubsequently transferred onto a nitrocellulose membrane (Whatman GmbH,Dassel, Germany). After blocking in 5% skim milk in PBS, the membranewas incubated with anti-beta-glucuronidase rabbit polyclonal antibody(G5420, Sigma-Aldrich, Schnelldorf, Germany), anti-beta-galactosidaserabbit polyclonal antibody (A-11132, Molecular Probes, Leiden,Netherlands), anti-GFP rabbit polyclonal antibody (sc-8334, Santa Cruz,Heidelberg, Germany) or anti-beta-actin mouse monoclonal antibody(ab6276, Abcam, Cambridge, UK). The first antibodies were detected usinghorseradish peroxidase-conjugated anti-mouse (ab6728, Abcam, Cambridge,UK) or anti-rabbit (ab6721, Abcam, Cambridge, UK) secondary antibodies,followed by enhanced chemiluminescence detection.

X-Gal/X-GlcU Staining and Microscopy Studies

For the analysis of expression and activity of beta-galactosidase andbeta-glucuronidase respectively, A549 cells were seeded on coverslipsand infected with 200 pfu (plaque forming units) GLV-1h68, rVACV-LacZ⁻or rVACV-GusA⁻ per well. After incubation for 2 days, cells were fixedusing 4% paraformaldehyde and washed twice in PBS. Staining solutionscontained of 40 μl X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside,Invitrogen, Karlsruhe, Germany) and X-GlcU(5-bromo-4-chloro-3-indolyl-β-D-glucuronide, Invitrogen, Karlsruhe,Germany) respectively in dimethylformamide (40 mg ml-1), ferricyanide(12 mM K₃Fe(CN)₆), 5.2 mM MgCl₂ and ferrocyanide solution (12 mMK₄Fe(CN)₆). Coverslips were stained with either X-Gal or X-GlcU solutionand incubated for 24 h at 37° C. before mounting in Mowiol. Images weretaken with a Zeiss Axiovert 200M microscope.

Histology and Immunofluorescence

For histological analysis, snap-frozen tumors were fixed in 4%paraformaldehyde/PBS overnight at 4° C. Samples were embedded in 5%(w/v) low-melt agarose (AppliChem, Darmstadt, Germany) and 100 μmsections were cut using a Leica VT1000S Vibratome (Leica, Heerbrugg,Switzerland) as described before (Stritzker et al., (2007) Int J MedMicrobiol 297-151:162). After permeabilizing in 0.3% Triton X-100/PBS,sections were incubated with Hoechst 33342, anti-beta-glucuronidaserabbit polyclonal antibody (G5420, Sigma-Aldrich, Schnelldorf, Germany)and anti-beta-galactosidase chicken polyclonal antibody (ab9361, Abcam,Cambridge, UK) before staining with Cy-5-conjugated donkey anti-rabbitand Cy-3 conjugated donkey anti-chicken secondary antibodies (JacksonImmunoResearch, West Grove, Pa.). Mowiol-embedded sections were examinedusing a Leica MZ 16 FA Stereo-Fluorescence Microscope equipped with aLeica DC500 Digital Camera. Digital Images were processed with Photoshop7.0 (Adobe Systems, San Jose, Calif.) and merged to yield pseudocoloredpictures.

Generation of Xenograft Tumors in Mice—Animal Studies

A549 and MTH52c xenograft tumors were developed in 6- to 8-week-old nudemice (NCI:Hsd:Athymic Nude FoxnI^(nu), Harlan Borchem, Germany) byimplanting 5×10⁶ cells subcutaneously in the right abdominal flank. Twoto three weeks after implantation, tumor bearing mice were anesthetizedwith isoflurane and injected i.v. with either PBS or virus, as describedbelow. Blood and urine collection of mice was carried out underanesthesia by a heparinised capillary pipet (No. 554/20, Assistent,Sondheim, Germany) via the retro-orbital sinus vein and a bladdercatheter (No. 381312, Becton Dickinson, Heidelberg, Germany) for bloodand urine respectively.

All animal experiments were carried out in accordance with protocolsapproved by the Regierung von Unterfranken (Würzburg, Germany) (protocolnumber AZ 55.2-2531.01-17/08) and/or the Institutional Animal Care andUse Committee (IACUC) of Explora BIOLABS, located in San Diego ScienceCenter (San Diego, USA) (protocol number: EB08-003).

Fluorogenic Probes and Detection of Fluorescence Products

The lyophilized fluorogenic probes fluorescein di-beta-D-glucuronide(FDGlcU), Fluorescein di-β-D-galactopyranoside (FDG) and4-Methylumbelliferyl-b-D-glucuronide (4-MUG) (Invitrogen, Karlsruhe,Germany) were dissolved in DMSO (36.5 mM). For in vivo studies, 5 μL ofeach stock dilution was mixed with 195 μL PBS and injectedintraperitoneally. Whole body and urine fluorescence analysis wasperformed using a Maestro EX imaging system (CRI, Woburn, Mass.). Forserum analysis, the collected mouse serum was diluted 1:15 with PBS and80 μL of each sample were mixed with of either 2.5 μg FDGlcU or 1.5 μg4-MUG if not otherwise indicated. Human serum of healthy individuals(Zen-Bio Inc, Research Triangle, N.C.) was obtained from whole blood and10 μL were used in the described assay. After incubation for 1 h at 37°C. (if not otherwise indicated), fluorescence was read in Lumox 384-wellplates (Sarstedt, Nümbrecht, Germany) using an Infinite 200 ProMicroplate Reader (Tecan, Crailsheim, Germany) or a Spectra Max M5(Molecular Devices, Sunnyvale, USA) and fluorescence intensities arelisted as relative fluorescence units.

Example 3 Fluorogenic Compound Activation in rVACV-Colonized Tumors

In this example, the activation and pharmacokinetics ofbeta-galactosidase and glucuronidase substrates, Fluoresceindi-β-D-galactopyranoside (FDG; substrate for the beta-galactosidaseLacZ; Invitrogen) and fluorescein di-beta-D-glucuronide (FDGlcU; aglucuronidase substrate; Invitrogen) in rVACV-colonized tumors wasdetermined.

FDG or FDGlcU (5 μL of 36.5 mM stock solution in 195 μL PBS) wereintraperitoneally injected into tumor bearing mice that had previouslybeen injected via retro-orbital sinus vein with 5×10⁶ pfu of oncolyticrVACV (GLV-1h68) encoding β-galactosidase and β-glucuronidase. Animalsthat were previously injected with PBS or 5×10⁶ pfu of control rVACVstrains not expressing β-galactosidase (rVACV-LacZ⁻) and β-glucuronidase(rVACV-GusA⁻) respectively served as controls. Fluorescein, whichresulted upon cleavage of either substrate, was detected using a smallanimal fluorescence imaging system.

The results show that FDG and FDGlcU were activated in the tumor, andactivation was dependent on the expression of LacZ and GusArespectively. As shown in FIG. 1, maximum fluorescence in the tumor wasobserved 120 min after intraperitoneal injection. As shown in FIG. 2,maximum fluorescence upon intravenous FDGlcU-injection was observed 20min post injection. Further, about 6 hours post injection (hpi), theGFP-dependent fluorescence remained while most of the compound specificfluorescence was gone.

Example 4 Analysis of Urine Samples from FDGlcU Injected Mice

In this example, urine was examined for the presence of fluoresceinresulting from cleavage of fluorogenic substrates by GLV-1h68. Theresults show that fluorescein was detected in the urine of micepreviously injected with GLV-1h68. Additionally, direct injection offluorescein into the tumor resulted in an accumulation of fluorescein inthe bladder, and subsequent secretion of the fluorescein with the urine,in addition to the disappearance of fluorescein from the tumor.

The presence of the fluorescein in the urine of GLV-1h68 injected tumorbearing mice was evaluated as a biomarker for successful tumorcolonization by the virus. Mice that were injected with either PBS(control, non-colonized) or with GusA-negative control-virus (GLV-1h188)or GusA-positive (GLV-1h68) were anesthetized and urine was isolated viaa bladder catheter before and 90 minutes after i.p. injection of FDGlcU.Urine fluorescence analysis on 5 μL of urine was performed using aMaestro EX imaging system (CRI, Woburn, Mass.).

As shown in FIG. 3, fluorescein was observed in the urine of GLV-1h68treated tumor bearing animals but was not observed in FDGlcU injectedmice that had either non-colonized or rVACV-GusA⁻ colonized tumors.Thus, the presence of GLV-1h68 in tumors of live mice was determinedwith a simple urine test after systemic injection of FDGlcU.

Example 5 Evaluation of β-Glucuronidase Specific Fluorogenic CompoundActivation in Serum of A549 Tumor Bearing Mice A. β-GlucuronidaseSpecific Fluorogenic Compound Activation in Serum of Tumor Bearing Mice

In this example, the presence of active β-glucuronidase in the serum ofGLV-1h68 injected tumor bearing mice was determined by 1) addition offluorogenic compound FDGlcU or 4-Methylumbelliferyl-b-D-glucuronide(4-MUG; Invitrogen) to the serum; and 2) detection of fluorescence. Tothis end, the 5 μL serum of tumor bearing mice that were previouslyinjected with GLV-1h68 was diluted with PBS to 75 μL and was incubatedwith either 2.5 μg FDGlcU or 1.5 μg 4-MUG (diluted in 5 μL), both ofwhich are hydrolyzed by β-glucuronidase to the fluorescent productsfluorescein and 4-methylumbelliferone (4-MU) respectively. Serum oftumor bearing mice that were injected with either PBS or a GusA-negativecontrol rVACV were used as negative controls. Fluorescence wasdetermined as described in Example 2 and was reported as relativefluorescence units (RFU). 4-MUG was determined at an excitationwavelength of 365(9) nm and an emission wavelength of 455(20) nm. FDGlcUwas determined at an excitation wavelength of 489(9) nm and an emissionwavelength of 520(20) nm.

The results show that no fluorescence was observed when mice wereinjected with either PBS or GusA-negative control rVACV whilefluorescence was detected for both fluorogenic compounds when mice wereinjected with GusA-positive GLV-1h68 (see FIG. 4A). Beta-glucuronidaseis not secreted (no secretion signal) after production. In order to getout of the cells and into the serum, the host cell must lyse.Subsequently, the enzyme is shed to the serum. Thus, the serum containedactive (non-secreted, but shed) enzymes that were produced in the tumortissues.

B. β-Glucuronidase Specific Fluorogenic Compound Activation in Serum ofTumor Bearing Mice

In this example, the assay was used to confirm the applicability of themethod using a larger number of samples and different tumor models.Serum samples (n=99) that were previously collected over a 4 year periodof time from different mouse tumor xenograft models, including thosecontaining GI-101A, A549, DU-145, PANC-1 and HT-29 tumors, were analyzedfor β-glucuronidase activity. The mice had previously been injected withPBS (n=33) or had previously been injected at 5×10⁶ pfu with severalGusA-positive (n=53, GLV-1h68, GLV-1h80 (derived from GLV-1h68containing MCP-1 gene instead of lacZ) or GusA-negative (n=13, GLV-1h90,GLV-1h43) rVACV strains virus for different periods of time (from 7 to53 days). The serum was diluted 1:15 with PBS and 75 μL of each samplewere mixed with either 2.5 μg FDGlcU or 1.5 μg 4-MUG (both in 5 μL) andfluorescence was determined as described in Example 2 and was reportedas relative fluorescence units (RFU).

As shown in FIG. 4B, a significant (p<0.001) difference was observedbetween the serum from tumor bearing mice treated with GusA-positiverVACV strains (average 4-MU or fluorescein in RFU of approximately 1×10⁴or 1×10⁵, respectively) and control mice that were treated withGusA-negative rVACV strains that do not express β-glucuronidase or PBS(average 4-MU in RFU of approximately 1×10²).

Example 6 Blood Test to Determine Successful Tumor Colonization

In this example, the assay was demonstrated to be a way to determinetumor colonization of GusA-encoding oncolytic virus strains in mice.

Tumor bearing mice (n=6) were systemically injected with a low dose(1×10⁵ PFU) of GLV-1h68. This dose was selected as it has previouslybeen shown to result in colonization of some tumors, while other tumorsare not colonized. Serum was isolated 1, 3, 7, 10 and 14 days postinjection and fluorescence was determined as described above. Mice weresacrificed at day 14 and tumor colonization was tested by conventionalplaque assay.

After 14 days, sera from two mice (#482 and #486) generated high 4-MUfluorescence (RFU of approximately 50,000 and 10,000 respectively) whilethe sera of the remaining mice did not generate fluorescence whentested. As stated above, the dose of GLV-1h68 was selected as it haspreviously been shown to result in colonization of some tumors, whileother tumors are not colonized. Viral titer analysis revealed that onlythe same two mice (#482 and #486) had virus colonized tumors. Thus,there was positive correlation between the positive FDGlcU/4-MUG basedblood tests and virus colonized tumors.

Example 7 Suitability of the Blood Test for Glucuronidase Activity toDifferentiate Between Tumor Bearing and Tumor Free Mice

In this example, the suitability of the blood test to differentiatebetween tumor bearing and control tumor free mice is shown. In addition,background levels of glucuronidase activity were determined.

a. GLV-1h68 Induced Glucuronidase Activity in Tumor Versus Tumor-FreeMice

Tumor bearing mice (n=5, Mice #7-11) and non-tumor bearing mice (n=6,Mice #1-6) were injected with 5×10⁶ pfu of GLV-1h68 in the retro-orbitalsinus vein. Seven (7) days post infection blood was drawn and serum wastested for glucuronidase activity upon the addition of 4-MUG or FDGlcU.

Analysis of sera revealed conversion of the fluorogenic compounds FDGlcUand 4-MUG in all tumor bearing mice seven days post infection. Low butevident glucuronidase activity was also detected in the serum ofnon-tumor bearing mice after seven days post infection. Closerexamination of the non-tumor bearing mice revealed GFP expression in thepaws of 2 mice (mice #1 and #4).

The mice were sacrificed on day 14 and a conventional plaque assay ofseveral organs was used to find the origin of glucuronidase production.The results of the plaque assay are set forth in Table 5 below. Apartfrom the two infected paws, virus was reproducibly isolated in lowconcentration from ovaries of non-tumor bearing mice (mice #1-#5). Incontrast, ovaries of tumor bearing mice were essentially free of virus.The data show significant viral distribution in the tumors of mice #7 to#11.

TABLE 5 Viral distribution in tumor bearing and non-tumor bearing mice14 days post infection pfu/g tissue fluorescence mouse # tumor bloodovaries spleen kidneys liver lung brain paw 4-MU fluorescein 1 NA ND NDND ND ND ND ND 2.10E+06 8599 1179 2 NA ND 2500 ND ND ND ND 100 ND 4418607 3 NA ND 5300 ND ND ND ND  20 ND 6775 855 4 NA ND 12000  ND 100 ND NDND 5.00E+05 14587 2641 5 NA ND 8800 ND ND 100 ND ND ND 4325 483 6 NA NDND ND ND  0 ND ND ND 5272 667 7 2.70E+07 ND ND 40 ND ND 20 ND ND 40812168977 8 5.15E+07 ND ND 100  ND ND ND ND ND 43732 61137 9 2.65E+07 ND NDND 100 ND ND ND ND 43866 43572 10 9.00E+06 ND  20 20 100 ND 80 ND ND38449 135454 11 8.50E+06 ND ND ND  20 ND ND ND ND 41645 28754 NA—notapplicable. ND—not detectable. Detection limit 20 pfu/g.

B. Background Expression of Glucuronidase Activity

Time course studies were performed in male (n=12 tumor bearing and 12tumor free) and female mice (n=24 tumor bearing and 6 tumor free)injected with 5×10⁶ pfu GLV-1h68. Blood was collected every other dayover a period of 14-16 days (in one half of the mice blood was taken oneven days, in the other half, blood was taken on uneven days post virusinjection). 4-MUG or FDGlcU were added and the serum was examined forglucuronidase activity by measuring the fluorescence as described inExample 2.

Glucuronidase activity was present in the serum of tumor-bearing mice.The results also showed low levels of glucuronidase present in the serumof tumor free mice. The results for non-tumor bearing mice were similarto those observed in tumor bearing mice until 8 days post virusinjection, after which time changes were observed between the twogroups. At day 8, the glucuronidase activity in the serum of tumor freemice decreased while the glucuronidase activity in the serum of tumorbearing mice increased, as observed by an increase in RFU. Significantdifferences (p<0.05) were detected between tumor bearing and non-tumorbeating mice after 9 days post infection. Taken together, 9 days afterinjection of the virus, it was possible to determine with confidencewhether A) an existing tumor was successfully colonized and/or B) atumor was present in the gusA encoding rVACV injected mouse, asevidenced by the presence of β-glucuronidase activity in serum samples.

Example 8 Assay Sensitivity

In this example, sensitivity of the assay was determined using in vitrostudies that measured the correlation between fluorescence signalintensity and increasing glucuronidase concentration, fluorogenicsubstrate concentration and incubation time. In addition, the effect ofthe presence of human serum on the assay was evaluated.

A. Assay Sensitivity

Helix pomatia glucuronidase (Sigma Aldrich, #G0751; 0, 0.049, 0.098,0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25.0, 50.0 and 100.0units; unit definition according to Sigma Aldrich: One Sigma or modified“Fishman” unit will liberate 1.0 μg of phenolphthalein fromphenolphthalein glucuronide per hr at 37° C. at pH 5.0; pH 6.8 for theE. coli source; 30 min assay) was incubated with fluorogenic substrate(4-MUG: 0, 0.094, 0.188, 0.375, 0.75, 1.5, 3, 6 and 12 μg; or FDGlcU: 0,0.156, 0.312, 0.625, 1.25, 2.5, 5, 10 and 20 μg) for an incubation timeof 15, 30, 60, 120 and 1080 minutes.

The results are set forth in FIG. 5 for substrate concentration (leftpanels) and incubation time (right panels). A positive correlation wasobserved between the fluorescence signal intensities and increasing A)glucuronidase concentration, B) substrate (FDGlcU or 4-MUG)concentration and C) incubation time. The data also revealed that verylow glucuronidase concentrations can be detected using the fluorogenicFDGlcU or 4-MUG substrates (0.156 and 0.094 units respectively). Thispermits detection of lysed tumor cells not only in mice but also inhumans.

B. Effect of Human Serum on Assay

The assay in the presence of human serum was shown by co-incubatingincreasing amounts of E. coli glucuronidase (0.000141, 0.000445,0.00141, 0.00445, 0.0141, 0.0445, 0.141, 0.445, 1.41, 4.45, 14.1, 44.5,445 ng) and either 4-MUG or FDGluC in the presence or absence of 10 μLhuman serum The data revealed that neither the sensitivity nor thefluorescence intensity of the assay was changed in the presence of humanserum.

Example 9 Minimal Amount of Infected Cancer Cells Required for PositiveDetection

This example shows the minimal amount of infected cancer cells requiredto generate a positive fluorescent signal, as the presence ofglucuronidase relies on the production by infected cancer cells.

A549 cells were infected at a multiplicity of infection of 2.0 ofGLV-1h68 or control-rVACV (rVACV-GusA^(neg)). One day later, the numberof infected cells was determined by counting and flow cytometry.Subsequently, the cells were diluted and seeded in half-log dilutions in384-well plates with concentrations varying from approximately 1.0 to1000 infected cells/well and co-incubated with 6.3 μg FDGlcU and 3.4 μg4-MUG respectively. To obtain high sensitivity, the probes wereincubated at 37° C. overnight and analyzed the next day.

The results show that a single cancer cell infected with GLV-1h68 can bedetected in the assay when using 4-MUG as a substrate (see FIG. 6). ForFDGlcU as a substrate, approximately 10 infected cancer cells wererequired to distinguish signal over background.

The sensitivity of the described test for the detection of tumors inhuman patients was determined using two different approaches: 1)Assuming that a greater volume of serum (e.g. 50 μL) is used whentesting the system on human patient samples, and considering an averagetotal blood volume of about 4.7 liters, approximately 10⁵ infectedcancer cells is sufficient to generate a detectable fluorescent signal.2) The fluorescent signal generated from a single cancer cell wassimilar to that obtained from 0.2 units glucuronidase (see Example 8).Increasing the sensitivity by adding more fluorescent substrate resultedin the detection of 0.05 units glucuronidase. This corresponds to aconcentration of 1 unit glucuronidase per mL serum (again using 50 μLserum per test) or 4700 units/average blood volume of a human patient.Therefore, as low as 2.4×10⁴ infected cancer cells are sufficient for apositive signal.

Example 10 Other Tumor Colonizing Vectors

In this example, the assay was performed using other exemplary tumorcolonizing vectors, namely E. coli Nissle 1917 xpBR322DEST_(inv)-P_(S10)-gusA-luxABCDE (EcN-SgusAL) and E. coli Nissle1917 x pBR322DEST_(inv)-P_(S10)-luxABCDE-term (EcN-SLT).

E. coli Nissle 1917 x pBR322DEST_(inv)-P_(S10)-gusA-luxABCDE(EcN-SgusAL) is an E. coli strain Nissle 1917 harboring a plasmidencoding β-glucuronidase and lux operon under a Bacillus subtilis rpsJpromoter (P_(S10); GenBank Accession No. U43929; SEQ ID NO:125). E. coliNissle 1917 x pBR322DEST_(inv)-P_(S10)-luxABCDE-term (EcN-SLT) is an E.coli strain Nissle 1917 harboring a plasmid the lux operon under controlof the Bacillus subtilis rpsJ promoter. This bacteria is a controlbacteria that does not express β-glucuronidase.

E. coli Nissle 1917 x pBR322DEST_(inv)-P_(S10)-gusA-luxABCDE(EcN-SgusAL) or E. coli Nissle 1917 xpBR322DEST_(inv)-P_(S10)-luxABCDE-term (EcN-SLT) bacteria or PBS wereintratumorally (i.t.) injected 2 days before serum analysis. Tumorcolonization was shown by bioluminescence imaging. In P_(S10)-gusAencoding E. coli Nissle 1917 injected mice, activation of FDGlcU and4-MUG was observed.

TABLE 6 T-test results for E. coli Nissle assay T-test Results FDGlcUdata EcN -SLT vs. EcN -SgusAL 0.165429222 EcN -SgusAL vs. PBS0.192366995 4-MUG data EcN -SLT vs. EcN -SgusAL 0.251668626 EcN -SgusALvs. PBS 0.24295584

Example 11 Analysis of Human Serum Samples from Individuals Treated withGLV-1h68

In this example, to monitor therapy, serum samples from cancer patientstreated with GLV-1h68 were examined for β-glucuronidase activity.

Patients were treated for one to six 28 day cycles, with intravenousadministration at day 1 of each cycle with GLV-1h68 in an amount between1×10⁵ to 3×10⁹ pfu. Samples were collected at various time points, from30 minutes to hours, to days. Various serum samples (20 μL) wereincubated with 3.75 μg 4-MUG for one hour. Fluorescence was determinedusing a SpectraMax M5 fluorometer and was reported as relativefluorescence units (RFU). The data show β-glucuronidase activity waspresent in the serum of 8 of 12 patients tested, indicating that viruscolonized tumors and were replicating. Table 7 below indicatesglucuronidase activity in picograms. The different cohorts receivedvarying amounts of GLV-1h68 as follows: Cohort 1, 2×10⁵ pfu; Cohort 2,1×10⁶ pfu; Cohort 3, 1×10⁷ pfu; Cohort 4, 1×10⁸ pfu; Cohort 5, 1×10⁹pfu; Cohort 5a, 1×10⁹ pfu; Cohort 5b, 3×10⁹ pfu; Cohort 6, 5×10⁷ pfu;and Cohort 7, 5×10⁸ pfu. The results indicate the highest levels ofglucuronidase activity were detected in serum on cycle 1, day 8 and day9.

TABLE 7 Glucuronidase activity in human serum samples Cycle 1 2 3 4 5 6Day Cohort Screen 1 2 3 4 8 9 15 22 1 2 3 4 8 15 22 1 8 15 22 1 15 22 11 1 P102 0 1 0 0 0 0 0 0 0 0 1 P103 3 3 4 4 4 6 5 5 4 1 P201 0 0 0 1 0 00 0 0 0 2 P202 0 0 0 0 0 0 0 0 0 0 2 P104 0 0 0 0 0 0 0 0 0 2 P105 0 0 00 0 0 0 0 3 P106 0 0 2 245 3 P204 1 0 4 2 2 2 3 3 2 2 3 1 2 3 P109 0 1 00 0 0 0 0 0 0 4 P205 0 0 1 0 3 0 6 5 0 2 4 P111 0 0 0 0 0 0 0 1 2 4 P1120 0 0 1 0 1 1 4 1 0 0 5 P208 0 0 0 0 0 0 0 0 0 0 0 0 0 5 P114 0 0 4 54 10 0 0 0 0 0 0 0 1 0 0 0 5 P116 0 0 0 91 157 0 0 0 0 0 0 0 0 0 5a P121 00 2 0 0 5a P213 1 2 6 3 3 5 0 5a P215 5b P120 0 0 0 0 0 0 0 0 0 0 5bP122 0 0 0 0 0 0 0 0 0 0 5b P216 4 9 1 17 6 P209 0 0 0 0 0 0 0 0 0 0 6P117 0 0 0 1 0 0 0 6 P212 0 0 0 0 0 0 2 1 3 2 7 P119 1 0 0 5 6 3 1 0 0 04 0 7 P124 0 0 0 0 0 0 0 0 0 0 7 P125 0 0 0 0 0 0 0 0 0

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A method of detecting replication in or colonization of a targetlocus in a subject by a therapeutic bacterium or detecting replicationof the therapeutic bacterium in a non-target cell in the subject,comprising: obtaining a sample from a subject to whom the therapeutichas been administered; testing the sample to detect a shed proteinencoded by the bacterium, wherein: the therapeutic bacterium accumulatesin tumors, tumor tissues, metastases, areas of inflammation,immunoprivileged sites or tissues, wounds or infections; a target locusis tumor, metastasis, inflamed tissue or wounded tissue; and detectionof a shed protein encoded by the bacterium indicates that the bacteriumis replicating in and/or colonizing a target locus.
 2. The method ofclaim 1, wherein the bacterium is a non-pathogenic mutual, commensual orprobiotic strain of bacteria.
 3. The method of claim 1, wherein thebacterium is selected from among Escherichia coli, Bacteroides,Eubacterium, Streptococcus, Actinomyces, Veillonella, Nesseria,Prevotella, Campylobacter, Fusobacterium, Eikenella, Porphyromonas,Priopionibacteria, Clostridia, Salmonella, Shigella, Bifidobacteria andStaphylococcus species.
 4. The method of claim 1, wherein the bacteriumis an Escherichia coli Nissle strain bacterium.
 5. A method formonitoring therapeutic progress in a subject having tumors, comprising:(a) administering to the subject a bacterium encoding a β-glucuronidase;(b) obtaining a sample from the subject, wherein the sample is a bodyfluid or tissue sample that is not a tumor sample; (c) detectingβ-glucuronidase activity in the sample, wherein the detection is by theaddition of a substrate for β-glucuronidase; and (d) determining thepresence of a product catalyzed by the reaction of the β-glucuronidasewith the substrate, wherein the detection of the product indicates thatthe bacterium has colonized or is replicating in a tumor tissue or cellin the subject and is treating the tumor such that therapy should becontinued.
 6. The method of claim 5, wherein the bacterium is anon-pathogenic mutual, commensual or probiotic strain of bacteria. 7.The method of claim 5, wherein the bacterium is selected from amongEscherichia coli, Bacteroides, Eubacterium, Streptococcus, Actinomyces,Veillonella, Nesseria, Prevotella, Campylobacter, Fusobacterium,Eikenella, Porphyromonas, Priopionibacteria, Clostridia, Salmonella,Shigella, Bifidobacteria and Staphylococcus species.
 8. The method ofclaim 5, wherein the bacterium is an Escherichia coli Nissle strainbacterium.
 9. The method of claim 5, wherein the β-glucuronidase is ahuman or bacterial β-glucuronidase.
 10. The method of claim 9, whereinthe β-glucuronidase comprises the sequence of amino acids set forth inSEQ ID NO:121, or a catalytically active portion thereof, or a sequenceof amino acids having at least 85% sequence identity with the sequenceset forth in SEQ ID NO:121.
 11. The method of claim 9, wherein theβ-glucuronidase comprises the sequence of amino acids set forth in SEQID NO:4, or a catalytically active portion thereof, or a sequence ofamino acids having at least 85% sequence identity with the sequence setforth in SEQ ID NO:4.
 12. The method of claim 9, wherein theβ-glucuronidase comprises the sequence of amino acids set forth in SEQID NO:4, or a catalytically active portion thereof, or a sequence ofamino acids having at least 90% sequence identity with the sequence setforth in SEQ ID NO:4.
 13. The method of claim 9, wherein theβ-glucuronidase comprises the sequence of amino acids set forth in anyof SEQ ID NOS:4, 114-121, 128, 130, 132, 134, 136, 138, 140, 142, 144and 146, or a catalytically active portion thereof, or a sequence ofamino acids having at least 85% sequence identity to the sequence setforth in any of SEQ ID NOS:4, 114-121, 128, 130, 132, 134, 136, 138,140, 142, 144 and
 146. 14. The method of claim 5, wherein the substrateis selected from the group consisting of fluorescein di-β-D-glucuronide(FDGlcU), 4-methylumbelliferyl-β-D-glucuronide (4-MUG),carboxyumbelliferyl β-D-glucuronide (CUGlcU),5-(pentafluorobenzoylamino)-fluorescein di-β-D-glucuronide (PFB-FDGlcU),C₁₂-fluorescein β-D-glucuronidase, 5-bromo-4-chloro-3-indolylβ-D-glucuronide (X-GlcU or BCIG), p-nitrophenyl-β-D-glucuronide,red-β-D-GlcU,CHA (magenta-β-D-GlcA;5-bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt),rose-β-D-GlcU,CHA (salmon-β-D-GlcUA;5-bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt),phenyl-β-D-glucuronide, and pharmaceutically acceptable salts thereof.15. The method of claim 14, wherein the substrate is selected from amongfluorescein di-β-D-glucuronide (FDGlcU) and4-methylumbelliferyl-β-D-glucuronide (4-MUG).
 16. The method of claim 5,wherein the sample is a body fluid that is selected from among blood,plasma, serum, lymph, ascetic fluid, cystic fluid, urine, nippleexudates, sweat, tears, saliva, mouth gargle, peritoneal fluid,cerebrospinal fluid (CSF), synovial fluid, aqueous humour, vitreoushumour, amniotic fluid, bile, cerumen (earwax), Cowper's fluid(pre-ejaculatory fluid), Chyle, Chyme, female ejaculate, interstitialfluid, lymph fluid, menses, breast milk, mucus, snot, phlegm, pleuralfluid, pus, sebum, semen, vaginal lubrication, and feces.
 17. The methodof claim 16, wherein the sample is collected between or between about 12hours and 1 month after treatment with the bacterium.
 18. The method ofclaim 16, wherein the sample is obtained within 1 week of treatment withthe virus, and detection of the β-glucuronidase activity in the sampleindicates that the virus is replicating in tumor cells and is effectivefor treatment.
 19. The method of claim 16, wherein the sample isobtained periodically following administration of the virus to monitorthe progress of treatment by detecting an increase in the amount ofβ-glucuronidase in the sample, indicating replication of the virus intumors, followed by a decrease indicating that tumors are shrinking. 20.The method of claim 5, wherein the cancer comprises a bladder tumor,breast tumor, prostate tumor, glioma tumor, adenocarcinoma, ovariancarcinoma, pancreatic carcinoma, liver tumor, skin tumor, pancreaticcancer, non-small cell lung cancer, multiple myeloma, leukemia, lung andbronchus tumor, breast tumor, colon and rectum tumor, kidney tumor,stomach tumor, esophagus tumor, liver and intrahepatic bile duct tumor,urinary bladder tumor, brain tumor and other nervous system tumor, headand neck tumor, oral cavity tumor, pharynx tumor, cervix tumor, uterinecorpus tumor, thyroid tumor, ovary tumor, testes tumor, prostate tumor,malignant melanoma, cholangiocarcinoma, thymoma, non-melanoma skincancer, hematologic tumor, malignancy, childhood leukemia and lymphoma,multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic andcutaneous origin, acute and chronic leukemia, acute lymphoblasticleukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasmacell neoplasm, lymphoid neoplasm or is a cancer associated with HIVinfection.
 21. The method of claim 5, wherein the cancer is a solidtumor.
 22. The method of claim 5, wherein the β-glucuronidase is asecreted protein.
 23. The method of claim 5, wherein the β-glucuronidaseis shed from infected cells.
 24. The method of claim 5, wherein theβ-glucuronidase is heterologous to the bacterium.
 25. The method ofclaim 1, wherein the shed protein is an endogenous protein.
 26. Themethod of claim 1, wherein the shed protein is β-glucuronidase.
 27. Themethod of claim 1, wherein the shed protein is heterologous to thebacterium.